RESUMO
Fatty acids are essential cellular building blocks and a major energy source. Regardless of their metabolic fate, fatty acids first need to be activated by forming a thioester with a coenzyme A group. This reaction is carried out by acyl-CoA synthetases (ACSs), of which ACSL1 (long-chain acyl-CoA synthetase 1) is an important member. Two bacterial homologues of ACSL1 crystal structures have been solved previously. One is a soluble dimeric protein, and the other is a monomeric peripheral membrane protein. The mammalian ACSL1 is a membrane protein with an N-terminal transmembrane helix. To characterize the mammalian ACSL1, we purified the full-length mouse ACSL1 and reconstituted it into lipid nanodiscs. Using enzymatic assays, mutational analysis, and cryo-electron microscopy, we show that mouse ACSL1 is active as a monomer.
Assuntos
Coenzima A Ligases/química , Animais , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Cristalografia por Raios X , Camundongos , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
BACKGROUND: Blood-brain barrier (BBB) dysfunction/disruption followed by leukocyte infiltration into the brain causes neuroinflammation and contributes to morbidity in multiple sclerosis, encephalitis, traumatic brain injury, and stroke. The identification of pathways that decreases the inflammatory potential of leukocytes would prevent such injury. Poly(ADP-ribose) polymerase 1 (PARP) controls various genes via its interaction with myriad transcription factors. Selective PARP inhibitors have appeared lately as potent anti-inflammatory tools. Their effects are outside the recognized PARP functions in DNA repair and transcriptional regulation. In this study, we explored the idea that selective inhibition of PARP in leukocytes would diminish their engagement of the brain endothelium. METHODS: Cerebral vascular changes and leukocyte-endothelium interactions were surveyed by intravital videomicroscopy utilizing a novel in vivo model of localized aseptic meningitis when TNFα was introduced intracerebrally in wild-type (PARP+/+) and PARP-deficient (PARP-/-) mice. The effects of selective PARP inhibition on primary human monocytes ability to adhere to or migrate across the BBB were also tested in vitro, employing primary human brain microvascular endothelial cells (BMVEC) as an in vitro model of the BBB. RESULTS: PARP suppression in monocytes diminished their adhesion to and migration across BBB in vitro models and prevented barrier injury. In monocytes, PARP inactivation decreased conformational activation of integrins that plays a key role in their tissue infiltration. Such changes were mediated by suppression of activation of small Rho GTPases and cytoskeletal rearrangements in monocytes. In vitro observations were confirmed in vivo showing diminished leukocyte-endothelial interaction after selective PARP suppression in leukocytes accompanied by BBB protection. PARP knockout animals demonstrated a substantial diminution of inflammatory responses in brain microvasculature and a decrease in BBB permeability. CONCLUSIONS: These results suggest PARP inhibition in leukocytes as a novel approach to BBB protection in the setting of endothelial dysfunction caused by inflammation-induced leukocyte engagement.
RESUMO
BACKGROUND: Universal access to the School Breakfast Program (SBP) is intended to help low-income and food-insecure students overcome barriers to eating breakfast. However, SBP participation is often still low despite universal access. Further information is needed with regard to these children's breakfast behaviors, and in particular breakfast behaviors among youth from food-insecure families, to inform effective breakfast interventions. OBJECTIVES: The objective of this study was to examine breakfast behaviors among a large sample of urban students with universal access to the SBP and to identify differences in breakfast behaviors among children from food-secure compared with food-insecure households. METHODS: A cross-sectional study of 821 fourth- through sixth-grade students and their parents from 16 schools was conducted. Students reported the foods/drinks selected and location of obtaining food/drink on the morning of data collection, parents reported household food security status using the 6-item Food Security Survey Module, and the school district provided SBP participation data during the fall semester of 2013. Multivariable linear regression models accounting for school-level clustering were used to examine differences in breakfast behaviors across 3 levels of household food security: food secure, low food secure, and very low food secure. RESULTS: Students participated in the SBP 31.2% of possible days, with 13% never participating in the SBP. One-fifth (19.4%) of students purchased something from a corner store for breakfast, and 16.9% skipped breakfast. Forty-six percent of students were food insecure; few differences in breakfast behaviors were observed across levels of food security. CONCLUSIONS: Despite universal access to the SBP, participation in the SBP is low. Breakfast skipping and selection of foods of low nutritional quality in the morning are common, regardless of household food security status. Additional novel implementation of the SBP and addressing students' breakfast preferences may be necessary to further reduce barriers to students obtaining a free, healthful breakfast. This trial was registered at clinicaltrials.gov as NCT01924130.
Assuntos
Desjejum , Comportamento Alimentar , Abastecimento de Alimentos , Valor Nutritivo , Pobreza , População Urbana , Peso Corporal , Criança , Fenômenos Fisiológicos da Nutrição Infantil , Comportamento de Escolha , Estudos Transversais , Feminino , Preferências Alimentares , Humanos , Modelos Lineares , Masculino , Análise Multivariada , Instituições AcadêmicasRESUMO
Cannabinoid receptor 2 (CB2) is highly expressed in immune cells and stimulation decreases inflammatory responses. We tested the idea that selective CB2 activation in human monocytes suppresses their ability to engage the brain endothelium and migrate across the blood-brain barrier (BBB), preventing consequent injury. Intravital videomicroscopy was used to quantify adhesion of leukocytes to cortical vessels in lipopolysaccharide-induced neuroinflammation, after injection of ex vivo CB2-activated leukocytes into mice; CB2 agonists markedly decreased adhesion of ex vivo labeled cells in vivo. In an in vitro BBB model, CB2 activation in monocytes largely attenuated adhesion to and migration across monolayers of primary human brain microvascular endothelial cells and diminished BBB damage. CB2 stimulation in monocytes down-regulated active forms of integrins, lymphocyte function-associated antigen 1 (LFA-1), and very late antigen 4 (VLA-4). Cells treated with CB2 agonists exhibited increased phosphorylation levels of inhibitory sites of the actin-binding proteins cofilin and VASP, which are upstream regulators of conformational integrin changes. Up-regulated by relevant stimuli, Rac1 and RhoA were suppressed by CB2 agonists in monocytes. CB2 stimulation decreased formation of lamellipodia, which play a key role in monocyte migration. These results indicate that selective CB2 activation in leukocytes decreases key steps in monocyte-BBB engagement, thus suppressing inflammatory leukocyte responses and preventing neuroinflammation.
Assuntos
Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Endotélio/metabolismo , Leucócitos/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Animais , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/patologia , Encefalite/metabolismo , Encefalite/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio/patologia , Humanos , Integrina alfa4beta1/química , Integrina alfa4beta1/metabolismo , Integrina beta1/metabolismo , Lipopolissacarídeos , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Microvasos/patologia , Monócitos/metabolismo , Monócitos/patologia , Fosfoproteínas/metabolismo , Fosforilação , Pseudópodes/metabolismo , Receptor CB2 de Canabinoide/agonistas , Migração Transendotelial e Transepitelial , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Previous studies have shown that modulation of the receptor-mediated cannabinoid system during neuroinflammation can produce potent neuroprotective and anti-inflammatory effects. However, in this context, little is known about how selective activation of the cannabinoid type-2 receptor (CB2R) affects the activated state of the brain endothelium and blood-brain barrier (BBB) function. Using human brain tissues and primary human brain microvascular endothelial cells (BMVECs), we demonstrate that the CB2R is highly upregulated during inflammatory insult. We then examined whether the CB2R agonists could attenuate inflammatory responses at the BBB using a mouse model of LPS-induced encephalitis and highly selective CB2R agonists. Visualization by intravital microscopy revealed that administration of JWH133 [(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran] or a novel resorcinol-based compound, O-1966 (1-[4-(1,1-dimethyl-heptyl)-2,6-dimethoxy-phenyl]-3-methyl-cyclohexanol), greatly attenuated leukocyte adhesion in surface pial vessels and in deep ascending cortical postcapillary venules. BBB permeability assessments with small and large fluorescent tracers showed that CB2R agonists were effective at preventing barrier leakiness after LPS administration. To determine whether the effects by CB2R agonists on barrier protection are not only due to the CB2R modulation of immune cell function, we tested the agonists in vitro with barrier-forming primary BMVECs. Remarkably, the addition of CB2R agonist increased transendothelial electrical resistance and increased the amount of tight junction protein present in membrane fractions. Furthermore, CB2R agonists decreased the induction of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 surface expression in BMVECs exposed to various proinflammatory mediators. Together, these results suggest that pharmacological CB2R ligands offer a new strategy for BBB protection during neuroinflammation.
Assuntos
Barreira Hematoencefálica/fisiopatologia , Encefalite/patologia , Células Endoteliais/fisiologia , Leucócitos/fisiologia , Receptor CB2 de Canabinoide/metabolismo , Animais , Anisóis/farmacologia , Anisóis/uso terapêutico , Barreira Hematoencefálica/efeitos dos fármacos , Canfanos/farmacologia , Canabinoides/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Células Cultivadas , Cicloexanóis , Dextranos/metabolismo , Modelos Animais de Doenças , Impedância Elétrica , Encefalite/induzido quimicamente , Endotélio/metabolismo , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Lipopolissacarídeos/efeitos adversos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Pirazóis/farmacologia , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/deficiência , Estatísticas não Paramétricas , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Proteína da Zônula de Oclusão-1RESUMO
Glycogen synthase kinase (GSK) 3ß has been identified as a regulator of immune responses. We demonstrated previously that GSK3ß inhibition in human brain microvascular endothelial cells (BMVECs) reduced monocyte adhesion/migration across BMVEC monolayers. Herein, we tested the idea that GSK3ß inhibition in monocytes can diminish their ability to engage the brain endothelium and migrate across the blood-brain barrier. Pretreatment of primary monocytes with GSK3ß inhibitors resulted in a decrease in adhesion (60%) and migration (85%), with similar results in U937 monocytic cells. Monocyte-BMVEC interactions resulted in diminished barrier integrity that was reversed by GSK3ß suppression in monocytic cells. Because integrins mediate monocyte rolling/adhesion, we detected the active conformational form of very late antigen 4 after stimulation with a peptide mimicking monocyte engagement by vascular cell adhesion molecule-1. Peptide stimulation resulted in a 14- to 20-fold up-regulation of the active form of integrin in monocytes that was suppressed by GSK3ß inhibitors (40% to 60%). Because small GTPases, such as Rac1, control leukocyte movement, we measured active Rac1 after monocyte activation with relevant stimuli. Stimulation enhanced the level of active Rac1 that was diminished by GSK3ß inhibitors. Monocytes treated with GSK3ß inhibitors showed increased levels of inhibitory sites of the actin-binding protein, cofilin, and vasodilator-stimulated phosphoprotein-regulating conformational changes of integrins. These results indicate that GSK3ß inhibition in monocytes affects active integrin expression, cytoskeleton rearrangement, and adhesion via suppression of Rac1-diminishing inflammatory leukocyte responses.
Assuntos
Movimento Celular , Regulação para Baixo , Células Endoteliais/patologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Integrina alfa4beta1/química , Monócitos/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Complexo AIDS Demência/patologia , Fatores de Despolimerização de Actina/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Encéfalo/patologia , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Ativação Enzimática/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Integrina alfa4beta1/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Proteínas dos Microfilamentos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Migração Transendotelial e Transepitelial/efeitos dos fármacosRESUMO
While TXNIP (thioredoxin interacting protein) in the plasma membrane and vesicular location is known to negatively regulate cellular glucose uptake by facilitating glucose transporter endocytosis, the function of TXNIP in the nucleus is far less understood. Herein, we sought to determine the function of nuclear TXNIP in vivo, using a new HA-tagged TXNIP knock-in mouse model. We observed that TXNIP can be found in the nucleus of a variety of cells from different tissues including hepatocytes (liver), enterocytes (small intestine), exocrine cells (pancreas), and brown adipocytes (BAT). Further investigations into the role of nuclear TXNIP in BAT revealed that cold stress rapidly and transiently activated HSF1 (heat shock factor 1). HSF1 interaction with TXNIP during its activation is required for optimal HSF1 directed cold shock response in BAT.
RESUMO
Brown adipose tissue (BAT) has the ability to burn calories as heat. Utilizing BAT thermogenesis is thus an attractive way to combat obesity. However, the transcriptional network resulting in the lipid synthesis to oxidation shift during thermogenesis is not completely understood. Here, we report the regulation of two master regulators of adipogenesis, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα), during acute cold stress in BAT. We found PPARγ dissociates from DNA in a fifth of its binding sites and these include Cebpa enhancers, leading to decreased C/EBPα expression. This dissociation requires PPARγ binding to activating ligands and is thus modulated by diet. Meanwhile, PPARα also detaches from DNA, and co-activator PGC1α associates with ERRα as part of a transcriptional network regulating lipid metabolism. Subsequent global replacement of C/EBPα by C/EBPß and its associated transcriptional machinery is required for upregulation of structural lipid synthesis despite general upregulation of fatty acid oxidation.
RESUMO
Obese individuals experience low grade inflammation initiated within their adipose tissue. However, the early events that lead to the release of these inflammatory factors from adipose tissue are poorly characterized. To separate glucose effects from lipid effects on adipose tissue, we used an adipose-specific TXNIP knockout model where excess basal glucose influx into adipocytes led to modest increase in adiposity without using high fat diet. We found an uncoupling of two events that are generally presumed to be coregulated: (1) an increase of adipose tissue macrophage (ATM) number; and (2) pro-inflammatory activation of ATMs. These two events are associated with different triggering signals: elevated free fatty acids output and extracellular matrix remodeling with increased ATM number, whereas decreased adiponectin level with activated ATM. This separation reflects non-overlapping pathways regulated by glucose and lipids in adipocytes, and neither group alone is sufficient to elicit the full inflammatory response in adipose tissue.
RESUMO
Methamphetamine (METH) abuse is known to be associated with an inordinate rate of infections. Although many studies have described the association of METH exposure and immunosuppression, so far the underlying mechanism still remains elusive. In this study, we present evidence that METH exposure resulted in mitochondrial oxidative damage and caused dysfunction of primary human T cells. METH treatment of T lymphocytes led to a rise in intracellular calcium levels that enhanced the generation of reactive oxygen species. TCR-CD28 linked calcium mobilization and subsequent uptake by mitochondria in METH-treated T cells correlated with an increase in mitochondrion-derived superoxide. Exposure to METH-induced mitochondrial dysfunction in the form of marked decrease in mitochondrial membrane potential, increased mitochondrial mass, enhanced protein nitrosylation and diminished protein levels of complexes I, III, and IV of the electron transport chain. These changes paralleled reduced IL-2 secretion and T cell proliferative responses after TCR-CD28 stimulation indicating impaired T cell function. Furthermore, antioxidants attenuated METH-induced mitochondrial damage by preserving the protein levels of mitochondrial complexes I, III, and IV. Altogether, our data indicate that METH can cause T cell dysfunction via induction of oxidative stress and mitochondrial injury as underlying mechanism of immune impairment secondary to METH abuse.
Assuntos
Imunossupressores/toxicidade , Metanfetamina/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Células Cultivadas , Estimulantes do Sistema Nervoso Central/toxicidade , Citosol/efeitos dos fármacos , Citosol/imunologia , Citosol/metabolismo , Relação Dose-Resposta Imunológica , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/imunologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/imunologia , Microscopia de Fluorescência , Mitocôndrias/patologia , Espécies Reativas de Oxigênio/metabolismo , Subpopulações de Linfócitos T/patologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Human immunodeficiency virus 1 (HIV-1) infection may result in activation of peripheral monocytes followed by their infiltration into the CNS, where the release of proinflammatory mediators causes neurologic disease. Previously, we detected high levels of soluble CD40 ligand (CD40L) in CSF and plasma of HIV-infected patients with cognitive impairment. We now show that CD40, a receptor for CD40L, is highly expressed in brain endothelial cells of patients affected by HIV-1 encephalitis (HIVE), suggesting an important role for the CD40/CD40L dyad in regulating blood-brain barrier (BBB) functions. This concept was further supported by in vitro experiments. Exposure of primary human brain microvascular endothelial cells (BMVECs) to CD40L upregulated the expression of adhesion molecules intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, which caused a fourfold increase in monocyte adhesion to BMVECs and stimulated migration across an in vitro BBB model. Investigations into the intracellular signaling pathways that govern these events revealed that cJUN-N-terminal kinase (JNK) is critical to CD40 activation in the BMVECs. CD40L induced activation of mixed-lineage-kinase-3 and JNK, leading to the subsequent activation of cJUN/AP-1 (activating-protein-1). JNK inhibition in the BMVECs prevented CD40L-mediated induction of adhesion molecules, monocyte adhesion, and transendothelial migration. These new findings support the concept that the CD40/CD40L dyad plays an important role in HIVE neuroinflammation.
Assuntos
Complexo AIDS Demência/imunologia , Barreira Hematoencefálica/imunologia , Antígenos CD40/imunologia , Inflamação/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Complexo AIDS Demência/metabolismo , Adulto , Análise de Variância , Barreira Hematoencefálica/metabolismo , Western Blotting , Antígenos CD40/metabolismo , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Ligante de CD40/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Citometria de Fluxo , Imunofluorescência , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Inflamação/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Molécula 1 de Adesão de Célula Vascular/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Immune mediators and leukocyte engagement of brain microvascular endothelial cells (BMVECs) contribute to blood-brain barrier impairment during neuroinflammation. Glycogen synthase kinase 3beta (GSK3beta) was recently identified as a potent regulator of immune responses in in vitro systems and animal models. However, the role of GSK3beta in regulation of immune endothelial functions remains undetermined. Here we evaluated the effect of GSK3beta inhibition on the regulation of inflammatory responses in BMVECs. A focused PCR gene array of 84 genes was performed to identify the cytokine and chemokine gene expression profile in tumor necrosis factor (TNF) alpha-stimulated BMVECs after GSK3beta inactivation by specific inhibitors. Fifteen of 39 genes induced by TNFalpha stimulation were down-regulated after GSK3beta inhibition. Genes known to contribute to neuroinflammation that were most negatively affected by GSK3beta inactivation included IP-10/CXCL10, MCP-1/CCL2, IL-8/CXCL8, RANTES/CCL5, and Groalpha/CXCL1. GSK3beta suppression resulted in diminished secretion of these proinflammatory mediators by inflamed BMVECs detected by ELISA. GSK3beta inhibition in BMVECs reduced adhesion molecule expression as well as monocyte adhesion to and migration across cytokine stimulated BMVEC monolayers. Interactions of monocytes with TNFalpha-activated BMVECs led to barrier disruption, and GSK3beta suppression in the endothelium restored barrier integrity. GSK3beta inhibition in vivo substantially decreased leukocyte adhesion to brain endothelium under inflammatory conditions. In summary, inhibition of GSK3beta emerges as an important target for stabilization of the blood-brain barrier in neuroinflammation.
Assuntos
Encéfalo/efeitos dos fármacos , Encefalite/patologia , Células Endoteliais/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Complexo AIDS Demência/tratamento farmacológico , Complexo AIDS Demência/metabolismo , Complexo AIDS Demência/patologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Estudos de Casos e Controles , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Encefalite/prevenção & controle , Células Endoteliais/imunologia , Glicogênio Sintase Quinase 3 beta , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Tiadiazóis/farmacologia , Tiadiazóis/uso terapêutico , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Ureia/análogos & derivados , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
GLUT1 is a major glucose facilitator expressed ubiquitously among tissues. Upregulation of its expression plays an important role in the development of many types of cancer and metabolic diseases. Thioredoxin-interacting protein (TXNIP) is an α-arrestin that acts as an adaptor for GLUT1 in clathrin-mediated endocytosis. It regulates cellular glucose uptake in response to both intracellular and extracellular signals via its control on GLUT1-4. In order to understand the interaction between GLUT1 and TXNIP, we generated GLUT1 lipid nanodiscs and carried out isothermal titration calorimetry and single-particle electron microscopy experiments. We found that GLUT1 lipid nanodiscs and TXNIP interact in a 1:1 ratio and that this interaction requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2).
Assuntos
Proteínas de Transporte/genética , Transportador de Glucose Tipo 1/genética , Lipídeos/genética , Fosfatidilinositol 4,5-Difosfato/química , Transporte Biológico/genética , Proteínas de Transporte/química , Clatrina/química , Endocitose/genética , Glucose/metabolismo , Transportador de Glucose Tipo 1/química , Humanos , Lipídeos/química , Fosfatidilinositol 4,5-Difosfato/genética , Transdução de SinaisRESUMO
Hyperglycemia affects over 400 million individuals worldwide. The detrimental health effects are well studied at the tissue level, but the in vivo effects at the organelle level are poorly understood. To establish such an in vivo model, we used mice lacking TXNIP, a negative regulator of glucose uptake. Examining mitochondrial function in brown adipose tissue, we find that TXNIP KO mice have a lower content of polyunsaturated fatty acids (PUFAs) in their membrane lipids, which affects mitochondrial integrity and electron transport chain efficiency and ultimately results in lower mitochondrial heat output. This phenotype can be rescued by a ketogenic diet, confirming the usefulness of this model and highlighting one facet of early cellular damage caused by excess glucose influx.
Assuntos
Tecido Adiposo Marrom/metabolismo , Carboidratos da Dieta/efeitos adversos , Mitocôndrias/metabolismo , Tecido Adiposo Marrom/ultraestrutura , Animais , Transporte Biológico/genética , Proteínas de Transporte/metabolismo , Dieta Cetogênica , Ácidos Graxos Insaturados/metabolismo , Regulação da Expressão Gênica , Lipidômica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/ultraestrutura , Termogênese/genética , Tiorredoxinas/metabolismoRESUMO
Growth factors, such as insulin, can induce both acute and long-term glucose uptake into cells. Apart from the rapid, insulin-induced fusion of glucose transporter (GLUT)4 storage vesicles with the cell surface that occurs in muscle and adipose tissues, the mechanism behind acute induction has been unclear in other systems. Thioredoxin interacting protein (TXNIP) has been shown to be a negative regulator of cellular glucose uptake. TXNIP is transcriptionally induced by glucose and reduces glucose influx by promoting GLUT1 endocytosis. Here, we report that TXNIP is a direct substrate of protein kinase B (AKT) and is responsible for mediating AKT-dependent acute glucose influx after growth factor stimulation. Furthermore, TXNIP functions as an adaptor for the basal endocytosis of GLUT4 in vivo, its absence allows excess glucose uptake in muscle and adipose tissues, causing hypoglycemia during fasting. Altogether, TXNIP serves as a key node of signal regulation and response for modulating glucose influx through GLUT1 and GLUT4.
Assuntos
Tecido Adiposo/metabolismo , Proteínas de Transporte/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Tiorredoxinas/metabolismo , Células 3T3-L1 , Animais , Proteínas de Transporte/genética , Endocitose , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/genética , Tiorredoxinas/genéticaRESUMO
Brain pericytes are uniquely positioned within the neurovascular unit to provide support to blood brain barrier (BBB) maintenance. Neurologic conditions, such as HIV-1-associated neurocognitive disorder, are associated with BBB compromise due to chronic inflammation. Little is known about pericyte dysfunction during HIV-1 infection. We found decreased expression of pericyte markers in human brains from HIV-1-infected patients (even those on antiretroviral therapy). Using primary human brain pericytes, we assessed expression of pericyte markers (α1-integrin, α-smooth muscle actin, platelet-derived growth factor-B receptor ß, CX-43) and found their downregulation after treatment with tumor necrosis factor-α (TNFα) or interleukin-1 ß (IL-1ß). Pericyte exposure to virus or cytokines resulted in decreased secretion of factors promoting BBB formation (angiopoietin-1, transforming growth factor-ß1) and mRNA for basement membrane components. TNFα and IL-1ß enhanced expression of adhesion molecules in pericytes paralleling increased monocyte adhesion to pericytes. Monocyte migration across BBB models composed of human brain endothelial cells and pericytes demonstrated a diminished rate in baseline migration compared to constructs composed only of brain endothelial cells. However, exposure to the relevant chemokine, CCL2, enhanced the magnitude of monocyte migration when compared to BBB models composed of brain endothelial cells only. These data suggest an important role of pericytes in BBB regulation in neuroinflammation.
Assuntos
Encéfalo/patologia , Neurite (Inflamação)/patologia , Pericitos/patologia , Adulto , Membrana Basal/metabolismo , Barreira Hematoencefálica/patologia , Adesão Celular , Quimiocinas/metabolismo , Doença Crônica , Citocinas/biossíntese , Células Endoteliais/patologia , Feminino , Infecções por HIV/patologia , HIV-1 , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/patologia , Cultura Primária de Células , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Multifactorial mechanisms comprising countless cellular factors and virus-encoded transactivators regulate the transcription of HIV-1 (HIV). Since poly(ADP-ribose) polymerase 1 (PARP-1) regulates numerous genes through its interaction with various transcription factors, inhibition of PARP-1 has surfaced recently as a powerful anti-inflammatory tool. We suggest a novel tactic to diminish HIV replication via PARP-1 inhibition in an in vitro model system, exploiting human primary monocyte-derived macrophages (MDM). PARP-1 inhibition was capable to lessen HIV replication in MDM by 60-80% after 7 days infection. Tat, tumor necrosis factor α (TNFα), and phorbol 12-myristate 13-acetate (PMA) are known triggers of the Long Terminal Repeat (LTR), which can switch virus replication. Tat overexpression in MDM transfected with an LTR reporter plasmid resulted in a 4.2-fold increase in LTR activation; PARP inhibition caused 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85-95%). PARP inhibition in MDM exhibited 90% diminution in NFκB activity (known to mediate TNFα- and PMA-induced HIV LTR activation). Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These discoveries suggest that inactivation of PARP suppresses HIV replication in MDM by via attenuation of LTR activation, NFκB suppression and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide an effective approach to management of HIV infection.
RESUMO
Blood-brain barrier (BBB) dysfunction seen in neuroinflammation contributes to mortality and morbidity in multiple sclerosis, encephalitis, traumatic brain injury, and stroke. Identification of molecular targets maintaining barrier function is of clinical relevance. We used a novel in vivo model of localized aseptic meningitis where tumor necrosis factor alpha (TNFα) was introduced intracerebrally and surveyed cerebral vascular changes and leukocyte-endothelium interactions by intravital videomicroscopy. Poly(ADP-ribose) polymerase-1 (PARP) inhibition significantly reduced leukocyte adhesion to and migration across brain endothelium in cortical microvessels. PARP inactivation diminished BBB permeability in an in vivo model of systemic inflammation. PARP suppression in primary human brain microvascular endothelial cells (BMVEC), an in vitro model of BBB, enhanced barrier integrity and augmented expression of tight junction proteins. PARP inhibition in BMVEC diminished human monocyte adhesion to TNFα-activated BMVEC (up to 65%) and migration (80-100%) across BBB models. PARP suppression decreased expression of adhesion molecules and decreased activity of GTPases (controlling BBB integrity and monocyte migration across the BBB). PARP inhibitors down-regulated expression of inflammatory genes and dampened secretion of pro-inflammatory factors increased by TNFα in BMVEC. These results point to PARP suppression as a novel approach to BBB protection in the setting of endothelial dysfunction caused by inflammation.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Meningite Asséptica/fisiopatologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/fisiologia , Barreira Hematoencefálica/fisiopatologia , Western Blotting , Permeabilidade Capilar/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/imunologia , Células Endoteliais , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Endotélio Vascular/fisiopatologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Humanos , Leucócitos/citologia , Masculino , Meningite Asséptica/imunologia , Meningite Asséptica/metabolismo , Camundongos Endogâmicos C57BL , Microscopia de Vídeo , Poli(ADP-Ribose) Polimerase-1 , Proteínas de Junções Íntimas/metabolismo , Migração Transendotelial e Transepitelial/efeitos dos fármacosRESUMO
Chronic neuroinflammatory disorders (such as HIV associated neurodegeneration) require treatment that decreases production of inflammatory factors by activated microglia and macrophages and protection of blood brain barrier (BBB) injury secondary to activation of brain endothelium. Cannabioid type 2 receptor (CB2) is highly expressed on macrophages and brain microvasular enndothelial cells (BMVEC) and is upregulated in inflammation and HIV infection. It has been shown that CB2 activation dampened inflammatory responses in macrophages and BMVEC. In this study, we assessed by PCR array the expression of a wide range of genes increased in macrophages and BMVEC in inflammation. TNFα treatment upregulated 33 genes in primary human BMVEC, and two highly selective CB2 agonists diminished expression of 31 and 32 genes. These results were confirmed by functional assays (BBB protection after inflammatory insult and decreased migration of monocytes across BMVEC monolayers after CB2 stimulation). Similarly, CB2 stimulation in primary human macrophages led to the suppression of 35 genes out of the 50 genes upregulated by LPS. Such changes in gene expression paralleled diminished secretion of proinflammatory factors. These results indicate the potential utility of CB2 agonists for the treatment of neuroinflammation.
Assuntos
Encéfalo/metabolismo , Endotélio/metabolismo , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Células Cultivadas , Endotélio/efeitos dos fármacos , Endotélio/imunologia , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Receptor CB2 de Canabinoide/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Pathologic conditions in the central nervous system, regardless of the underlying injury mechanism, show a certain level of blood-brain barrier (BBB) impairment. Endothelial dysfunction is the earliest event in the initiation of vascular damage caused by inflammation due to stroke, atherosclerosis, trauma, or brain infections. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators. The relationship between neuroinflammation and miRNA expression in brain endothelium remains unexplored. Previously, we showed the BBB-protective and anti-inflammatory effects of glycogen synthase kinase (GSK) 3ß inhibition in brain endothelium in in vitro and in vivo models of neuroinflammation. Using microarray screening, we identified miRNAs induced in primary human brain microvascular endothelial cells after exposure to the pro-inflammatory cytokine, tumor necrosis factor-α, with/out GSK3ß inhibition. Among the highly modified miRNAs, let-7 and miR-98 were predicted to target the inflammatory molecules, CCL2 and CCL5. Overexpression of let-7 and miR-98 in vitro and in vivo resulted in reduced leukocyte adhesion to and migration across endothelium, diminished expression of pro-inflammatory cytokines, and increased BBB tightness, attenuating barrier 'leakiness' in neuroinflammation conditions. For the first time, we showed that miRNAs could be used as a therapeutic tool to prevent the BBB dysfunction in neuroinflammation.