RESUMEN
The myelin sheath, which is wrapped around axons, is a lipid-enriched structure produced by mature oligodendrocytes. Disruption of the myelin sheath is observed in several neurological diseases, such as multiple sclerosis. A crucial component of myelin is sphingomyelin, levels of which can be increased by ABCA8, a member of the ATP-binding cassette transporter family. ABCA8 is highly expressed in the cerebellum, specifically in oligodendroglia. However, whether ABCA8 plays a role in myelination and mechanisms that would underlie this role remain unknown. Here, we found that the absence of Abca8b, a mouse ortholog of ABCA8, led to decreased numbers of cerebellar oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes in mice. We show that in oligodendrocytes, ABCA8 interacts with chondroitin sulfate proteoglycan 4 (CSPG4), a molecule essential for OPC proliferation, migration, and myelination. In the absence of Abca8b, localization of CSPG4 to the plasma membrane was decreased, contributing to reduced cerebellar CSPG4 expression. Cerebellar CSPG4+ OPCs were also diminished, leading to decreased mature myelinating oligodendrocyte numbers and cerebellar myelination levels in Abca8b-/- mice. In addition, electron microscopy analyses showed that the number of nonmyelinated cerebellar axons was increased, whereas cerebellar myelin thickness (g-ratio), myelin sheath periodicity, and axonal diameter were all decreased, indicative of disordered myelin ultrastructure. In line with disrupted cerebellar myelination, Abca8b-/- mice showed lower cerebellar conduction velocity and disturbed locomotion. In summary, ABCA8 modulates cerebellar myelination, in part through functional regulation of the ABCA8-interacting protein CSPG4. Our findings suggest that ABCA8 disruption may contribute to the pathophysiology of myelin disorders.
Asunto(s)
Células Precursoras de OligodendrocitosRESUMEN
Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered α-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.
Asunto(s)
Aterosclerosis/prevención & control , Furina/antagonistas & inhibidores , Placa Aterosclerótica/tratamiento farmacológico , alfa 1-Antitripsina/uso terapéutico , Animales , Aorta/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Arteria Carótida Común , Progresión de la Enfermedad , Evaluación Preclínica de Medicamentos , Inducción Enzimática/efectos de los fármacos , Furina/genética , Furina/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos/fisiología , Masculino , Metaloproteinasa 2 de la Matriz/análisis , Ratones , Ratones Endogámicos C57BL , Monocitos/fisiología , Placa Aterosclerótica/patología , Receptores de LDL/deficiencia , Túnica Íntima/efectos de los fármacos , Túnica Íntima/patología , Remodelación Vascular , alfa 1-Antitripsina/farmacologíaRESUMEN
Bile acids (BAs) are surfactant molecules that regulate the intestinal absorption of lipids. Thus, the modulation of BAs represents a potential therapy for nonalcoholic fatty liver disease (NAFLD), which is characterized by hepatic accumulation of fat and is a major cause of liver disease worldwide. Cyp8b1 is a critical modulator of the hydrophobicity index of the BA pool. As a therapeutic proof of concept, we aimed to determine the impact of Cyp8b1 inhibition in vivo on BA pool composition and as protection against NAFLD. Inhibition of Cyp8b1 expression in mice led to a remodeling of the BA pool, which altered its signaling properties and decreased intestinal fat absorption. In a model of cholesterol-induced NAFLD, Cyp8b1 knockdown significantly decreased steatosis and hepatic lipid content, which has been associated with an increase in fecal lipid and BA excretion. Moreover, inhibition of Cyp8b1 not only decreased hepatic lipid accumulation, but also resulted in the clearance of previously accumulated hepatic cholesterol, which led to a regression in hepatic steatosis. Taken together, our data demonstrate that Cyp8b1 inhibition is a viable therapeutic target of crucial interest for metabolic diseases, such as NAFLD.-Chevre, R., Trigueros-Motos, L., Castaño, D., Chua, T., Corlianò, M., Patankar, J. V., Sng, L., Sim, L., Juin, T. L., Carissimo, G., Ng, L. F. P., Yi, C. N. J., Eliathamby, C. C., Groen, A. K., Hayden, M. R., Singaraja, R. R. Therapeutic modulation of the bile acid pool by Cyp8b1 knockdown protects against nonalcoholic fatty liver disease in mice.
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Ácidos y Sales Biliares/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Esteroide 12-alfa-Hidroxilasa/genética , Animales , Femenino , Células HEK293 , Humanos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/terapia , Tratamiento con ARN de Interferencia , Esteroide 12-alfa-Hidroxilasa/metabolismoRESUMEN
OBJECTIVE: High-density lipoproteins (HDL) are considered to protect against atherosclerosis in part by facilitating the removal of cholesterol from peripheral tissues. However, factors regulating lipid efflux are incompletely understood. We previously identified a variant in adenosine triphosphate-binding cassette transporter A8 (ABCA8) in an individual with low HDL cholesterol (HDLc). Here, we investigate the role of ABCA8 in cholesterol efflux and in regulating HDLc levels. APPROACH AND RESULTS: We sequenced ABCA8 in individuals with low and high HDLc and identified, exclusively in low HDLc probands, 3 predicted deleterious heterozygous ABCA8 mutations (p.Pro609Arg [P609R], IVS17-2 A>G and p.Thr741Stop [T741X]). HDLc levels were lower in heterozygous mutation carriers compared with first-degree family controls (0.86±0.34 versus 1.17±0.26 mmol/L; P=0.005). HDLc levels were significantly decreased by 29% (P=0.01) in Abca8b-/- mice on a high-cholesterol diet compared with wild-type mice, whereas hepatic overexpression of human ABCA8 in mice resulted in significant increases in plasma HDLc and the first steps of macrophage-to-feces reverse cholesterol transport. Overexpression of wild-type but not mutant ABCA8 resulted in a significant increase (1.8-fold; P=0.01) of cholesterol efflux to apolipoprotein AI in vitro. ABCA8 colocalizes and interacts with adenosine triphosphate-binding cassette transporter A1 and further potentiates adenosine triphosphate-binding cassette transporter A1-mediated cholesterol efflux. CONCLUSIONS: ABCA8 facilitates cholesterol efflux and modulates HDLc levels in humans and mice.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Colesterol en la Dieta/sangre , HDL-Colesterol/sangre , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Adulto , Anciano , Animales , Apolipoproteína A-I/sangre , Apolipoproteína B-100/sangre , Transporte Biológico , Biomarcadores/sangre , Células COS , Estudios de Casos y Controles , Chlorocebus aethiops , Análisis Mutacional de ADN , Dieta Alta en Grasa , Heces/química , Femenino , Células HEK293 , Herencia , Heterocigoto , Humanos , Hígado/metabolismo , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Mutación , Linaje , Fenotipo , TransfecciónRESUMEN
Huntington disease (HD) is an adult-onset neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms that is caused by a CAG expansion in the HTT gene. Palmitoylation is the addition of saturated fatty acids to proteins by DHHC palmitoylacyl transferases. HTT is palmitoylated by huntingtin interacting proteins 14 and 14-like (HIP14 and HIP14L or ZDHHC17 and 13 respectively). Mutant HTT is less palmitoylated and this reduction of palmitoylation accelerates its aggregation and increases cellular toxicity. Mouse models deficient in either Hip14 (Hip14(-/-)) or Hip14l (Hip14l(-/-)) develop HD-like phenotypes. The biological function of HTT palmitoylation and the role that loss of HTT palmitoylation plays in the pathogenesis of HD are unknown. To address these questions mice deficient for both genes were created. Loss of Hip14 and Hip14l leads to early embryonic lethality at day embryonic day 10-11 due to failed chorioallantoic fusion. The chorion is thickened and disorganized and the allantois does not fuse correctly with the chorion and forms a balloon-like shape compared to Hip14l(-/-); Hip14(+/+) littermate control embryos. Interestingly, the Hip14(-/-) ; Hip14(-/-) embryos share many features with the Htt(-/-) embryos, including folding of the yolk sac, a bulb shaped allantois, and a thickened and disorganized chorion. This may be due to a decrease in HTT palmitoylation. In Hip14(-/-); Hip14l(-/-) mouse embryonic fibroblasts show a 25% decrease in HTT palmitoylation compared to wild type cells. This is the first description of a double PAT deficient mouse model where loss of a PAT or multiple PATs results in embryonic lethality in mammals. These results reinforce the physiological importance of palmitoylation during embryogenesis.
Asunto(s)
Aciltransferasas/metabolismo , Membrana Corioalantoides/embriología , Fusión de Membrana/genética , Placenta/embriología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Aciltransferasas/genética , Animales , Western Blotting , Femenino , Genotipo , Hibridación in Situ , Lipoilación , Fusión de Membrana/fisiología , Ratones , Ratones Noqueados , Embarazo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
HIP14 is the most highly conserved of 23 human palmitoyl acyltransferases (PATs) that catalyze the post-translational addition of palmitate to proteins, including huntingtin (HTT). HIP14 is dysfunctional in the presence of mutant HTT (mHTT), the causative gene for Huntington disease (HD), and we hypothesize that reduced palmitoylation of HTT and other HIP14 substrates contributes to the pathogenesis of the disease. Here we describe the yeast two-hybrid (Y2H) interactors of HIP14 in the first comprehensive study of interactors of a mammalian PAT. Unexpectedly, we discovered a highly significant overlap between HIP14 interactors and 370 published interactors of HTT, 4-fold greater than for control proteins (P = 8 × 10(-5)). Nearly half of the 36 shared interactors are already implicated in HD, supporting a direct link between HIP14 and the disease. The HIP14 Y2H interaction set is significantly enriched for palmitoylated proteins that are candidate substrates. We confirmed that three of them, GPM6A, and the Sprouty domain-containing proteins SPRED1 and SPRED3, are indeed palmitoylated by HIP14; the first enzyme known to palmitoylate these proteins. These novel substrates functions might be affected by reduced palmitoylation in HD. We also show that the vesicular cargo adapter optineurin, an established HTT-binding protein, co-immunoprecipitates with HIP14 but is not palmitoylated. mHTT leads to mislocalization of optineurin and aberrant cargo trafficking. Therefore, it is possible that optineurin regulates trafficking of HIP14 to its substrates. Taken together, our data raise the possibility that defective palmitoylation by HIP14 might be an important mechanism that contributes to the pathogenesis of HD.
Asunto(s)
Aciltransferasas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Enfermedad de Huntington/genética , Proteínas del Tejido Nervioso/genética , Procesamiento Proteico-Postraduccional , Aciltransferasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células COS , Proteínas de Ciclo Celular , Chlorocebus aethiops , Redes Reguladoras de Genes , Células HEK293 , Humanos , Proteína Huntingtina , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipoilación , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Anotación de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Palmitoylation of neurotransmitter receptors and associated scaffold proteins regulates their membrane association in a rapid, reversible, and activity-dependent fashion. This makes palmitoylation an attractive candidate as a key regulator of the fast, reversible, and activity-dependent insertion of synaptic proteins required during the induction and expression of long-term plasticity. Here we describe that the constitutive loss of huntingtin interacting protein 14 (Hip14, also known as DHHC17), a single member of the broad palmitoyl acyltransferase (PAT) family, produces marked alterations in synaptic function in varied brain regions and significantly impairs hippocampal memory and synaptic plasticity. The data presented suggest that, even though the substrate pool is overlapping for the 23 known PAT family members, the function of a single PAT has marked effects upon physiology and cognition. Moreover, an improved understanding of the role of PATs in synaptic modification and maintenance highlights a potential strategy for intervention against early cognitive impairments in neurodegenerative disease.
Asunto(s)
Aciltransferasas/genética , Potenciación a Largo Plazo/genética , Potenciación a Largo Plazo/fisiología , Trastornos de la Memoria/genética , Plasticidad Neuronal/genética , Sinapsis/genética , Aciltransferasas/metabolismo , Análisis de Varianza , Animales , Recuento de Células , Dendritas/ultraestructura , Hipocampo/citología , Hipocampo/fisiología , Lipoilación , Ratones , Ratones Noqueados , Plasticidad Neuronal/fisiología , Técnicas de Placa-Clamp , Sinapsis/fisiologíaRESUMEN
A low level of HDL cholesterol (HDL-C) is a common clinical scenario and an important marker for increased cardiovascular risk. Many patients with very low or very high HDL-C have a rare mutation in one of several genes, but identification of the molecular abnormality in patients with extreme HDL-C is rarely performed in clinical practice. We investigated the accuracy and diagnostic yield of a targeted next-generation sequencing (NGS) assay for extreme levels of HDL-C. We developed a targeted NGS panel to capture the exons, intron/exon boundaries, and untranslated regions of 26 genes with highly penetrant effects on plasma lipid levels. We sequenced 141 patients with extreme HDL-C levels and prioritized variants in accordance with medical genetics guidelines. We identified 35 pathogenic and probably pathogenic variants in HDL genes, including 21 novel variants, and performed functional validation on a subset of these. Overall, a molecular diagnosis was established in 35.9% of patients with low HDL-C and 5.2% with high HDL-C, and all prioritized variants identified by NGS were confirmed by Sanger sequencing. Our results suggest that a molecular diagnosis can be identified in a substantial proportion of patients with low HDL-C using targeted NGS.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/genética , Enfermedades Cardiovasculares/genética , HDL-Colesterol/sangre , HDL-Colesterol/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Hipercolesterolemia/sangre , Hipercolesterolemia/genética , Transportador 1 de Casete de Unión a ATP/sangre , Alelos , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/diagnóstico , Exones , Femenino , Estudios de Asociación Genética , Humanos , Intrones , Masculino , Persona de Mediana Edad , Factores de RiesgoRESUMEN
Palmitoylation, the dynamic post-translational addition of the lipid, palmitate, to proteins by Asp-His-His-Cys-containing palmitoyl acyltransferase (PAT) enzymes, modulates protein function and localization and plays a key role in the nervous system. Huntingtin-interacting protein 14 (HIP14), a well-characterized neuronal PAT, has been implicated in the pathogenesis of Huntington disease (HD), a fatal neurodegenerative disease associated with motor, psychiatric and cognitive symptoms, caused by a CAG expansion in the huntingtin gene (HTT). Mice deficient for Hip14 expression develop neuropathological and behavioural features similar to HD, and the catalytic activity of HIP14 is impaired in HD mice, most likely due to the reduced interaction of HIP14 with HTT. Huntingtin-interacting protein 14-like (HIP14L) is a paralog of HIP14, with identical domain structure. Together, HIP14 and HIP14L are the major PATs for HTT. Here, we report the characterization of a Hip14l-deficient mouse model, which develops adult-onset, widespread and progressive neuropathology accompanied by early motor deficits in climbing, impaired motor learning and reduced palmitoylation of a novel HIP14L substrate: SNAP25. Although the phenotype resembles that of the Hip14(-/-) mice, a more progressive phenotype, similar to that of the YAC128 transgenic mouse model of HD, is observed. In addition, HIP14L interacts less with mutant HTT than the wild-type protein, suggesting that reduced HIP14L-dependent palmitoylation of neuronal substrates may contribute to the pathogenesis of HD. Thus, both HIP14 and HIP14L may be dysfunctional in the disease.
Asunto(s)
Aciltransferasas/genética , Modelos Animales de Enfermedad , Eliminación de Gen , Enfermedad de Huntington/genética , Neuronas/patología , Aciltransferasas/deficiencia , Aciltransferasas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Femenino , Regulación de la Expresión Génica , Proteína Huntingtina , Enfermedad de Huntington/patología , Immunoblotting , Aprendizaje/fisiología , Lipoilación , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia de ADN , Proteína 25 Asociada a Sinaptosomas/genética , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
While genetic determinants strongly influence HDL cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family-based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, cholesteryl ester transfer protein (CETP), endothelial lipase (LIPG), and UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the proband's family. Family-based association analyses were performed for variants with sufficient power to detect significance at P < 0.05 with a total of 627 family members being assessed. Mutations in the genes glucokinase regulatory protein (GCKR), RNase L (RNASEL), leukocyte immunoglobulin-like receptor 3 (LILRA3), and dynein axonemal heavy chain 10 (DNAH10) segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in four novel genes that may play a role in regulating HDLc levels in humans.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Dineínas Axonemales/genética , HDL-Colesterol/sangre , Endorribonucleasas/genética , Mutación , Receptores Inmunológicos/genética , Transportador 1 de Casete de Unión a ATP/genética , Adulto , Anciano , Apolipoproteína A-I/genética , Proteínas de Transferencia de Ésteres de Colesterol/genética , HDL-Colesterol/genética , Femenino , Humanos , Lipasa/genética , Masculino , Persona de Mediana Edad , N-Acetilgalactosaminiltransferasas/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
Huntingtin interacting protein 14 (HIP14, ZDHHC17) is a huntingtin (HTT) interacting protein with palmitoyl transferase activity. In order to interrogate the function of Hip14, we generated mice with disruption in their Hip14 gene. Hip14-/- mice displayed behavioral, biochemical and neuropathological defects that are reminiscent of Huntington disease (HD). Palmitoylation of other HIP14 substrates, but not Htt, was reduced in the Hip14-/- mice. Hip14 is dysfunctional in the presence of mutant htt in the YAC128 mouse model of HD, suggesting that altered palmitoylation mediated by HIP14 may contribute to HD.
Asunto(s)
Aciltransferasas/deficiencia , Enfermedad de Huntington/etiología , Lipoilación/genética , Proteínas del Tejido Nervioso/deficiencia , Aciltransferasas/genética , Aciltransferasas/metabolismo , Animales , Muerte Celular/genética , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Encefalinas/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Ratones , Ratones Noqueados , Actividad Motora/genética , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/patología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Sinapsis/metabolismoRESUMEN
Familial exudative vitreoretinopathy (FEVR) is a hereditary ocular disorder characterized by a failure of peripheral retinal vascularization. Loci associated with FEVR map to 11q13-q23 (EVR1; OMIM 133780, ref. 1), Xp11.4 (EVR2; OMIM 305390, ref. 2) and 11p13-12 (EVR3; OMIM 605750, ref. 3). Here we have confirmed linkage to the 11q13-23 locus for autosomal dominant FEVR in one large multigenerational family and refined the disease locus to a genomic region spanning 1.55 Mb. Mutations in FZD4, encoding the putative Wnt receptor frizzled-4, segregated completely with affected individuals in the family and were detected in affected individuals from an additional unrelated family, but not in normal controls. FZD genes encode Wnt receptors, which are implicated in development and carcinogenesis. Injection of wildtype and mutated FZD4 into Xenopus laevis embryos revealed that wildtype, but not mutant, frizzled-4 activated calcium/calmodulin-dependent protein kinase II (CAMKII) and protein kinase C (PKC), components of the Wnt/Ca(2+) signaling pathway. In one of the mutants, altered subcellular trafficking led to defective signaling. These findings support a function for frizzled-4 in retinal angiogenesis and establish the first association between a Wnt receptor and human disease.
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Neovascularización Patológica/genética , Proteínas/genética , Enfermedades de la Retina/genética , Vasos Retinianos/patología , Secuencia de Aminoácidos , Preescolar , Femenino , Receptores Frizzled , Marcadores Genéticos , Haplotipos , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Linaje , Polimorfismo Genético , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Retina/patología , Enfermedades de la Retina/patología , Alineación de Secuencia , Transducción de SeñalRESUMEN
Intermittent fasting (IF) has been shown to reduce cardiovascular risk factors in both animals and humans, and can protect the heart against ischemic injury in models of myocardial infarction. However, the underlying molecular mechanisms behind these effects remain unclear. To shed light on the molecular and cellular adaptations of the heart to IF, we conducted comprehensive system-wide analyses of the proteome, phosphoproteome, and transcriptome, followed by functional analysis. Using advanced mass spectrometry, we profiled the proteome and phosphoproteome of heart tissues obtained from mice that were maintained on daily 12- or 16 hr fasting, every-other-day fasting, or ad libitum control feeding regimens for 6 months. We also performed RNA sequencing to evaluate whether the observed molecular responses to IF occur at the transcriptional or post-transcriptional levels. Our analyses revealed that IF significantly affected pathways that regulate cyclic GMP signaling, lipid and amino acid metabolism, cell adhesion, cell death, and inflammation. Furthermore, we found that the impact of IF on different metabolic processes varied depending on the length of the fasting regimen. Short IF regimens showed a higher correlation of pathway alteration, while longer IF regimens had an inverse correlation of metabolic processes such as fatty acid oxidation and immune processes. Additionally, functional echocardiographic analyses demonstrated that IF enhances stress-induced cardiac performance. Our systematic multi-omics study provides a molecular framework for understanding how IF impacts the heart's function and its vulnerability to injury and disease.
Asunto(s)
Ayuno Intermitente , Multiómica , Humanos , Ratones , Animales , Proteoma , Ayuno/fisiología , Metabolismo EnergéticoRESUMEN
Atherosclerosis is a progressive disease in which macrophages play an essential role. Macrophage infiltration into the arterial wall induces the development of an early atherosclerotic lesion. However, the dynamics of macrophage infiltration into the arterial wall during lesion progression remain poorly understood. In this study, low-density lipoprotein receptor knockout mice were fed a Western-type diet for 3, 6, 9, and 12 weeks to induce the formation of atherosclerotic lesions with different degrees of complexity. Subsequently, these mice underwent transplantation with bone marrow-overexpressing enhanced green fluorescent protein to track donor-derived cells, including macrophages. After 8 weeks of Western-type diet feeding after transplantation, macrophage infiltration was evaluated by immunohistochemical staining of donor-derived macrophages (enhanced green fluorescent protein-positive F4/80(+)) in the aortic roots. We found that the growth of pre-existing initial lesions was mainly caused by continued recruitment of donor-derived macrophages into the arterial wall. Interestingly, macrophage infiltration into pre-existing more advanced lesions was largely impaired, likely because of the formation of fibrous caps. In addition, interference with the expression of macrophage ATP-binding cassette transporter 1, an ATP-binding cassette transporter involved in cellular cholesterol efflux and macrophage recruitment into tissues, affects the infiltration of macrophages into pre-existing early lesions but not into advanced lesions. In conclusion, our data suggest that the dynamics of macrophage infiltration into the arterial wall vary greatly during atherogenesis and, thus, may affect the efficiency of pharmaceutical interventions aimed at targeting macrophage infiltration into the arterial wall.
Asunto(s)
Arterias/patología , Aterosclerosis/patología , Movimiento Celular , Macrófagos/patología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Animales , Aterosclerosis/genética , Trasplante de Médula Ósea , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Hígado/patología , Ganglios Linfáticos/patología , Macrófagos/fisiología , Ratones , Ratones Noqueados , Receptores de LDL/genética , Bazo/patologíaRESUMEN
Caffeine is among the most highly consumed substances worldwide, and it has been associated with decreased cardiovascular risk. Although caffeine has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), the mechanism underlying this effect is unknown. Here, we demonstrated that caffeine decreased VSMC proliferation and induced macroautophagy/autophagy in an in vivo vascular injury model of restenosis. Furthermore, we studied the effects of caffeine in primary human and mouse aortic VSMCs and immortalized mouse aortic VSMCs. Caffeine decreased cell proliferation, and induced autophagy flux via inhibition of MTOR signaling in these cells. Genetic deletion of the key autophagy gene Atg5, and the Sqstm1/p62 gene encoding a receptor protein, showed that the anti-proliferative effect by caffeine was dependent upon autophagy. Interestingly, caffeine also decreased WNT-signaling and the expression of two WNT target genes, Axin2 and Ccnd1 (cyclin D1). This effect was mediated by autophagic degradation of a key member of the WNT signaling cascade, DVL2, by caffeine to decrease WNT signaling and cell proliferation. SQSTM1/p62, MAP1LC3B-II and DVL2 were also shown to interact with each other, and the overexpression of DVL2 counteracted the inhibition of cell proliferation by caffeine. Taken together, our in vivo and in vitro findings demonstrated that caffeine reduced VSMC proliferation by inhibiting WNT signaling via stimulation of autophagy, thus reducing the vascular restenosis. Our findings suggest that caffeine and other autophagy-inducing drugs may represent novel cardiovascular therapeutic tools to protect against restenosis after angioplasty and/or stent placement.
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Autofagia , Músculo Liso Vascular , Animales , Autofagia/fisiología , Cafeína/metabolismo , Cafeína/farmacología , Proliferación Celular , Células Cultivadas , Humanos , Ratones , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteína Sequestosoma-1/metabolismo , Vía de Señalización WntRESUMEN
BACKGROUNDCytochrome P450 family 8 subfamily B member 1 (CYP8B1) generates 12α-hydroxylated bile acids (BAs) that are associated with insulin resistance in humans.METHODSTo determine whether reduced CYP8B1 activity improves insulin sensitivity, we sequenced CYP8B1 in individuals without diabetes and identified carriers of complete loss-of-function (CLOF) mutations utilizing functional assays.RESULTSMutation carriers had lower plasma 12α-hydroxylated/non-12α-hydroxylated BA and cholic acid (CA)/chenodeoxycholic acid (CDCA) ratios compared with age-, sex-, and BMI-matched controls. During insulin clamps, hepatic glucose production was suppressed to a similar magnitude by insulin, but glucose infusion rates to maintain euglycemia were higher in mutation carriers, indicating increased peripheral insulin sensitivity. Consistently, a polymorphic CLOF CYP8B1 mutation associated with lower fasting insulin in the AMP-T2D-GENES study. Exposure of primary human muscle cells to mutation-carrier CA/CDCA ratios demonstrated increased FOXO1 activity, and upregulation of both insulin signaling and glucose uptake, which were mediated by increased CDCA. Inhibition of FOXO1 attenuated the CDCA-mediated increase in muscle insulin signaling and glucose uptake. We found that reduced CYP8B1 activity associates with increased insulin sensitivity in humans.CONCLUSIONOur findings suggest that increased circulatory CDCA due to reduced CYP8B1 activity increases skeletal muscle insulin sensitivity, contributing to increased whole-body insulin sensitization.FUNDINGBiomedical Research Council/National Medical Research Council of Singapore.
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Resistencia a la Insulina , Esteroide 12-alfa-Hidroxilasa , Humanos , Esteroide 12-alfa-Hidroxilasa/genética , Resistencia a la Insulina/genética , Insulina/genética , Haploinsuficiencia , Ácidos y Sales Biliares , Ácido Cólico , GlucosaRESUMEN
Loss of ABCA1 activity in Tangier disease (TD) is associated with abnormal apoB lipoprotein (Lp) metabolism in addition to the complete absence of high density lipoprotein (HDL). We used hepatocyte-specific ABCA1 knock-out (HSKO) mice to test the hypothesis that hepatic ABCA1 plays dual roles in regulating Lp metabolism and nascent HDL formation. HSKO mice recapitulated the TD lipid phenotype with postprandial hypertriglyceridemia, markedly decreased LDL, and near absence of HDL. Triglyceride (TG) secretion was 2-fold higher in HSKO compared with wild type mice, primarily due to secretion of larger TG-enriched VLDL secondary to reduced hepatic phosphatidylinositol 3-kinase signaling. HSKO mice also displayed delayed clearance of postprandial TG and reduced post-heparin plasma lipolytic activity. In addition, hepatic LDLr expression and plasma LDL catabolism were increased 2-fold in HSKO compared with wild type mice. Last, adenoviral repletion of hepatic ABCA1 in HSKO mice normalized plasma VLDL TG and hepatic phosphatidylinositol 3-kinase signaling, with a partial recovery of HDL cholesterol levels, providing evidence that hepatic ABCA1 is involved in the reciprocal regulation of apoB Lp production and HDL formation. These findings suggest that altered apoB Lp metabolism in TD subjects may result from hepatic VLDL TG overproduction and increased hepatic LDLr expression and highlight hepatic ABCA1 as an important regulatory factor for apoB-containing Lp metabolism.
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/genética , Hepatocitos/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/biosíntesis , Triglicéridos/biosíntesis , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenoviridae/genética , Animales , Apolipoproteínas B/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Marcación de Gen , Humanos , Hipertrigliceridemia/genética , Hipertrigliceridemia/metabolismo , Lipoproteínas HDL/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Enfermedad de Tangier/genética , Enfermedad de Tangier/metabolismoRESUMEN
ATP-binding cassette transporter (ABC)A1 lipidates apolipoprotein A-I both directly at the plasma membrane and also uses lipids from the late endosomal or lysosomal compartment in the internal lipidation of apolipoprotein A-I. However, how ABCA1 targeting to these specific membranes is regulated remains unknown. Palmitoylation is a dynamically regulated lipid modification that targets many proteins to specific membrane domains. We hypothesized that palmitoylation may also regulate ABCA1 transport and function. Indeed, ABCA1 is robustly palmitoylated at cysteines 3, -23, -1110, and -1111. Abrogation of palmitoylation of ABCA1 by mutation of the cysteines results in a reduction of ABCA1 localization at the plasma membranes and a reduction in the ability of ABCA1 to efflux lipids to apolipoprotein A-I. ABCA1 is palmitoylated by the palmitoyl transferase DHHC8, and increasing DHHC8 protein results in increased ABCA1-mediated lipid efflux. Thus, palmitoylation regulates ABCA1 localization at the plasma membrane, and regulates its lipid efflux ability.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Procesamiento Proteico-Postraduccional , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Aciltransferasas/genética , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Apolipoproteína A-I/metabolismo , Transporte Biológico , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Colesterol/metabolismo , Cisteína , Humanos , Lipoilación , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Palmitatos/metabolismo , Fosfolípidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Relación Estructura-Actividad , TransfecciónRESUMEN
Bile acids are signalling hormones involved in the regulation of several metabolic pathways. The ability of bile acids to bind and signal through their receptors is modulated by the gut microbiome, since the microbiome contributes to the regulation and synthesis of bile acids as well to their physiochemical properties. From the gut, bacteria have been shown to send signals to the central nervous system via their metabolites, thus affecting the behaviour and brain function of the host organism. In the last years it has become increasingly evident that bile acids affect brain function, during normal physiological and pathological conditions. Although bile acids may be synthesized locally in the brain, the majority of brain bile acids are taken up from the systemic circulation. Since the composition of the brain bile acid pool may be regulated by the action of intestinal bacteria, it is possible that bile acids function as a communication bridge between the gut microbiome and the brain. However, little is known about the molecular mechanisms and the physiological roles of bile acids in the central nervous system. The possibility that bile acids may be a direct link between the intestinal microbiome and the brain is also an understudied subject. Here we review the influence of gut bacteria on the bile acid pool composition and properties, as well as striking evidence showing the role of bile acids as neuroactive molecules.
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Ácidos y Sales Biliares/metabolismo , Encéfalo/metabolismo , Microbioma Gastrointestinal , Animales , Colesterol/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Ingestión de Alimentos , Células Enterocromafines/metabolismo , Fermentación , Vesícula Biliar/metabolismo , Vida Libre de Gérmenes , Humanos , Hígado/metabolismo , Ratones , Enfermedades Neurodegenerativas/metabolismo , Neurotransmisores/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Accidente Cerebrovascular/metabolismo , Xantomatosis Cerebrotendinosa/metabolismoRESUMEN
Vascular restenosis remains a major problem in patients with coronary artery disease (CAD) and peripheral artery disease (PAD). Neointimal hyperplasia, defined by post-procedure proliferation and migration of vascular smooth muscle cells (VSMCs) is a key underlying pathology. Here we investigated the role of Interleukin 11 (IL-11) in a mouse model of injury-related plaque development. Apoe-/- mice were fed a hyperlipidaemic diet and subjected to carotid wire injury of the right carotid. Mice were injected with an anti-IL11 antibody (X203), IgG control antibody or buffer. We performed ultrasound analysis to assess vessel wall thickness and blood velocity. Using histology and immunofluorescence approaches, we determined the effects of IL-11 inhibition on VSMC and macrophages phenotypes and fibrosis. Treatment of mice with carotid wire injury using X203 significantly reduced post-endothelial injury vessel wall thickness, and injury-related plaque, when compared to control. Immunofluorescence staining of the injury-related plaque showed that X203 treatment did not reduce macrophage numbers, but reduced the number of VSMCs and lowered matrix metalloproteinase 2 (MMP2) levels and collagen content in comparison to control. X203 treatment was associated with a significant increase in smooth muscle protein 22α (SM22α) positive cells in injury-related plaque compared to control, suggesting preservation of the contractile VSMC phenotype. Interestingly, X203 also reduced the collagen content of uninjured carotid arteries as compared to IgG, showing an additional effect on hyperlipidemia-induced arterial remodeling in the absence of mechanical injury. Therapeutic inhibition of IL-11 reduced vessel wall thickness, attenuated neointimal hyperplasia, and has favorable effects on vascular remodeling following wire-induced endothelial injury. This suggests IL-11 inhibition as a potential novel therapeutic approach to reduce arterial stenosis following revascularization in CAD and PAD patients.