RESUMEN
Adenosine diphosphate ribosylation (ADP-ribosylation; ADPr), the addition of ADP-ribose moieties onto proteins and nucleic acids, is a highly conserved modification involved in a wide range of cellular functions, from viral defence, DNA damage response (DDR), metabolism, carcinogenesis and neurobiology. Here we study MACROD1 and MACROD2 (mono-ADP-ribosylhydrolases 1 and 2), two of the least well-understood ADPr-mono-hydrolases. MACROD1 has been reported to be largely localized to the mitochondria, while the MACROD2 genomic locus has been associated with various neurological conditions such as autism, attention deficit hyperactivity disorder (ADHD) and schizophrenia; yet the potential significance of disrupting these proteins in the context of mammalian behaviour is unknown. Therefore, here we analysed both Macrod1 and Macrod2 gene knockout (KO) mouse models in a battery of well-defined, spontaneous behavioural testing paradigms. Loss of Macrod1 resulted in a female-specific motor-coordination defect, whereas Macrod2 disruption was associated with hyperactivity that became more pronounced with age, in combination with a bradykinesia-like gait. These data reveal new insights into the importance of ADPr-mono-hydrolases in aspects of behaviour associated with both mitochondrial and neuropsychiatric disorders.
Asunto(s)
Conducta Animal , Hidrolasas de Éster Carboxílico/deficiencia , Enzimas Reparadoras del ADN/deficiencia , Hidrolasas/deficiencia , Animales , Peso Corporal , Hidrolasas de Éster Carboxílico/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Femenino , Marcha , Técnicas de Inactivación de Genes , Genotipo , Hidrolasas/metabolismo , Masculino , Ratones Noqueados , Actividad Motora , Reproducibilidad de los ResultadosRESUMEN
Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum. PA23 produces several inhibitory compounds that are under control of a complex regulatory network. Included in this cascade is the PhzRI quorum sensing (QS) system, which plays an essential role in PA23 biocontrol, as well as CsaRI and AurRI, which have not yet been characterized in PA23. The focus of the current study was to employ RNA sequencing to explore the spectrum of PA23 genes under QS control. In this work, we investigated genes under the control of the main QS transcriptional regulator, PhzR, as well as those differentially expressed in an AHL-deficient strain, PA23-6863, which constitutively expresses an AiiA lactonase, rendering the strain QS defective. Transcriptomic profiling revealed 545 differentially expressed genes (365 downregulated; 180 upregulated) in the phzR mutant and 534 genes (382 downregulated; 152 upregulated) in the AHL-deficient PA23-6863. In both strains, decreased expression of phenazine, pyrrolnitrin, and exoprotease biosynthetic genes was observed. We have previously reported that QS activates expression of these genes and their encoded products. In addition, elevated siderophore and decreased chitinase gene expression was observed in the QS-deficient stains, which was confirmed by phenotypic analysis. Inspection of the promoter regions revealed the presence of "phz-box" sequences in only 58 of the 807 differentially expressed genes, suggesting that much of the QS regulon is indirectly regulated. Consistent with this notion, 41 transcriptional regulators displayed altered expression in one or both of the QS-deficient strains. Collectively, our findings indicate that QS governs expression of approximately 13% of the PA23 genome affecting diverse functions ranging from secondary metabolite production to general metabolism.
Asunto(s)
Control Biológico de Vectores , Pseudomonas chlororaphis/genética , Percepción de Quorum/genética , Regulón/genética , Proteínas Bacterianas/genética , Hidrolasas de Éster Carboxílico/deficiencia , Movimiento Celular/genética , Quitinasas/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Mutantes , RNA-Seq , Sideróforos/genética , Transactivadores/genética , TranscriptomaRESUMEN
Corticotropin-releasing factor (CRF) regulates diverse physiological functions, including bladder control. We recently reported that Crf expression is under genetic control of Aoah, the locus encoding acyloxyacyl hydrolase (AOAH), suggesting that AOAH may also modulate voiding. Here, we examined the role of AOAH in bladder function. AOAH-deficient mice exhibited enlarged bladders relative to wild-type mice and had decreased voiding frequency and increased void volumes. AOAH-deficient mice had increased nonvoiding contractions and increased peak voiding pressure in awake cystometry. AOAH-deficient mice also exhibited increased bladder permeability and higher neuronal firing rates of bladder afferents in response to stretch. In wild-type mice, AOAH was expressed in bladder projecting neurons and colocalized in CRF-expressing neurons in Barrington's nucleus, an important brain area for voiding behavior, and Crf was elevated in Barrington's nucleus of AOAH-deficient mice. We had previously identified aryl hydrocarbon receptor (AhR) and peroxisome proliferator-activated receptor-γ as transcriptional regulators of Crf, and conditional knockout of AhR or peroxisome proliferator-activated receptor-γ in Crf-expressing cells restored normal voiding in AOAH-deficient mice. Finally, an AhR antagonist improved voiding in AOAH-deficient mice. Together, these data demonstrate that AOAH regulates bladder function and that the AOAH-Crf axis is a therapeutic target for treating voiding dysfunction.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Neuronas/enzimología , Vejiga Urinaria/inervación , Trastornos Urinarios/enzimología , Micción , Urodinámica , Animales , Compuestos Azo/farmacología , Núcleo de Barrington/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hidrolasas de Éster Carboxílico/deficiencia , Hidrolasas de Éster Carboxílico/genética , Hormona Liberadora de Corticotropina/metabolismo , Femenino , Masculino , Ratones Endogámicos C57BL , Contracción Muscular , Neuronas/efectos de los fármacos , PPAR gamma/genética , PPAR gamma/metabolismo , Presión , Pirazoles/farmacología , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Vejiga Urinaria/efectos de los fármacos , Micción/efectos de los fármacos , Trastornos Urinarios/tratamiento farmacológico , Trastornos Urinarios/genética , Trastornos Urinarios/fisiopatología , Urodinámica/efectos de los fármacosRESUMEN
A novel poly-3-hydroxybutyrate depolymerase was identified in Azotobacter vinelandii. This enzyme, now designated PhbZ1, is associated to the poly-3-hydroxybutyrate (PHB) granules and when expressed in Escherichia coli, it showed in vitro PHB depolymerizing activity on native or artificial PHB granules, but not on crystalline PHB. Native PHB (nPHB) granules isolated from a PhbZ1 mutant had a diminished endogenous in vitro hydrolysis of the polyester, when compared to the granules of the wild-type strain. This in vitro degradation was also tested in the presence of free coenzyme A. Thiolytic degradation of the polymer was observed in the nPHB granules of the wild type, resulting in the formation of 3-hydroxybutyryl-CoA, but was absent in the granules of the mutant. It was previously reported that cultures of A. vinelandii OP grown in a bioreactor showed a decrease in the weight average molecular weight (Mw) of the PHB after 20 h of culture, with an increase in the fraction of polymers of lower molecular weight. This decrease was correlated with an increase in the PHB depolymerase activity during the culture. Here, we show that in the phbZ1 mutant, neither the decrease in the Mw nor the appearance of a low molecular weight polymers occurred. In addition, a higher PHB accumulation was observed in the cultures of the phbZ1 mutant. These results suggest that PhbZ1 has a role in the degradation of PHB in cultures in bioreactors and its inactivation allows the production of a polymer of a uniform high molecular weight.
Asunto(s)
Azotobacter vinelandii/enzimología , Azotobacter vinelandii/metabolismo , Hidrolasas de Éster Carboxílico/deficiencia , Hidroxibutiratos/química , Hidroxibutiratos/metabolismo , Poliésteres/química , Poliésteres/metabolismo , Reactores Biológicos/microbiología , Hidrolasas de Éster Carboxílico/metabolismo , Eliminación de Gen , Peso MolecularRESUMEN
OBJECTIVE: To demonstrate that mutations in the phosphatidylglycerol remodelling enzyme SERAC1 can cause juvenile-onset complicated hereditary spastic paraplegia (cHSP) clusters, thus adding SERAC1 to the increasing number of complex lipid cHSP genes. METHODS: Combined genomic and functional validation studies (whole-exome sequencing, mRNA, cDNA and protein), biomarker investigations (3-methyl-glutaconic acid, filipin staining and phosphatidylglycerols PG34:1/PG36:1), and clinical and imaging phenotyping were performed in six affected subjects from two different branches of a large consanguineous family. RESULTS: 5 of 6 affected subjects shared cHSP as a common disease phenotype. Three subjects presented with juvenile-onset oligosystemic cHSP, still able to walk several miles at age >10-20 years. This benign phenotypic cluster and disease progression is strikingly divergent to the severe infantile phenotype of all SERAC1 cases reported so far. Two family members showed a more multisystemic juvenile-onset cHSP, indicating an intermediate phenotype between the benign oligosystemic cHSP and the classic infantile SERAC1 cluster. The homozygous splice mutation led to loss of the full-length SERAC1 protein and impaired phosphatidylglycerol PG34:1/PG36:1 remodelling. These phosphatidylglycerol changes, however, were milder than in classic infantile-onset SERAC1 cases, which might partially explain the milder SERAC1 phenotype. CONCLUSIONS: Our findings add SERAC1 to the increasing list of complex lipid cHSP genes. At the same time they redefine the phenotypic spectrum of SERAC1 deficiency. It is associated not only with the severe infantile-onset 'Methylglutaconic aciduria, Deafness, Encephalopathy, Leigh-like' syndrome (MEGDEL syndrome), but also with oligosystemic juvenile-onset cHSP as part of the now unfolding SERAC1 deficiency spectrum.
Asunto(s)
Hidrolasas de Éster Carboxílico/deficiencia , Mutación/genética , Sitios de Empalme de ARN/genética , Paraplejía Espástica Hereditaria/genética , Secuencia de Bases , Biomarcadores/metabolismo , Líquidos Corporales/metabolismo , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Familia , Femenino , Fibroblastos/metabolismo , Genómica , Homocigoto , Humanos , Intrones/genética , Imagen por Resonancia Magnética , Masculino , Linaje , Fenotipo , Fosfatidilgliceroles/metabolismoRESUMEN
Chronic pelvic pain causes significant patient morbidity and is a challenge to clinicians. Using a murine neurogenic cystitis model that recapitulates key aspects of interstitial cystitis/bladder pain syndrome (IC), we recently showed that pseudorabies virus (PRV) induces severe pelvic allodynia in BALB/c mice relative to C57BL/6 mice. Here, we report that a quantitative trait locus (QTL) analysis of PRV-induced allodynia in F2CxB progeny identified a polymorphism on chromosome 13, rs6314295 , significantly associated with allodynia (logarithm of odds = 3.11). The nearby gene encoding acyloxyacyl hydrolase ( Aoah) was induced in the sacral spinal cord of PRV-infected mice. AOAH-deficient mice exhibited increased vesicomotor reflex in response to bladder distension, consistent with spontaneous bladder hypersensitivity, and increased pelvic allodynia in neurogenic cystitis and postbacterial chronic pain models. AOAH deficiency resulted in greater bladder pathology and tumor necrosis factor production in PRV neurogenic cystitis, markers of increased bladder mast cell activation. AOAH immunoreactivity was detectable along the bladder-brain axis, including in brain sites previously correlated with human chronic pelvic pain. Finally, AOAH-deficient mice had significantly higher levels of bladder vascular endothelial growth factor, an emerging marker of chronic pelvic pain in humans. These findings indicate that AOAH modulates pelvic pain severity, suggesting that allelic variation in Aoah influences pelvic pain in IC.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Cistitis Intersticial/enzimología , Infecciones por Escherichia coli/enzimología , Hiperalgesia/enzimología , Dolor Pélvico/enzimología , Seudorrabia/enzimología , Vejiga Urinaria/inervación , Infecciones Urinarias/enzimología , Animales , Conducta Animal , Hidrolasas de Éster Carboxílico/deficiencia , Hidrolasas de Éster Carboxílico/genética , Cistitis Intersticial/genética , Cistitis Intersticial/fisiopatología , Cistitis Intersticial/psicología , Modelos Animales de Enfermedad , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/fisiopatología , Infecciones por Escherichia coli/psicología , Femenino , Predisposición Genética a la Enfermedad , Hiperalgesia/genética , Hiperalgesia/fisiopatología , Hiperalgesia/psicología , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Percepción del Dolor , Umbral del Dolor , Dolor Pélvico/genética , Dolor Pélvico/fisiopatología , Fenotipo , Seudorrabia/genética , Seudorrabia/fisiopatología , Seudorrabia/psicología , Sitios de Carácter Cuantitativo , Índice de Severidad de la Enfermedad , Factor de Necrosis Tumoral alfa/metabolismo , Vejiga Urinaria/metabolismo , Infecciones Urinarias/genética , Infecciones Urinarias/fisiopatología , Infecciones Urinarias/psicología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Ces1g/Es-x deficiency in mice results in weight gain, insulin resistance, fatty liver and hyperlipidemia through upregulation of de novo lipogenesis and oversecretion of triacylglycerol (TG)-rich lipoproteins. Here, we show that restoration of Ces1g/Es-x expression only in the liver significantly reduced hepatic TG concentration accompanied by decreased size of lipid droplets, reduced secretion of very low-density lipoproteins and improved insulin-mediated signal transduction in the liver. Collectively, these results demonstrate that hepatic Ces1g/Es-x plays a critical role in limiting hepatic steatosis, very low-density lipoprotein assembly and in augmenting insulin sensitivity.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Hígado Graso/prevención & control , Terapia Genética , Hiperlipidemias/prevención & control , Resistencia a la Insulina , Insulina/sangre , Metabolismo de los Lípidos , Hígado/enzimología , Animales , Glucemia/metabolismo , Hidrolasas de Éster Carboxílico/deficiencia , Hidrolasas de Éster Carboxílico/genética , Modelos Animales de Enfermedad , Hígado Graso/sangre , Hígado Graso/enzimología , Hígado Graso/genética , Femenino , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Predisposición Genética a la Enfermedad , Hiperlipidemias/sangre , Hiperlipidemias/enzimología , Hiperlipidemias/genética , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos/genética , Lipoproteínas VLDL/sangre , Ratones Noqueados , Fenotipo , Transducción de Señal , Factores de Tiempo , Triglicéridos/sangreRESUMEN
Muscle differentiation requires a complex signaling cascade that leads to the production of multinucleated myofibers. Genes regulating the intrinsic mitochondrial apoptotic pathway also function in controlling cell differentiation. How such signaling pathways are regulated during differentiation is not fully understood. Bit-1 (also known as PTRH2) mutations in humans cause infantile-onset multisystem disease with muscle weakness. We demonstrate here that Bit-1 controls skeletal myogenesis through a caspase-mediated signaling pathway. Bit-1-null mice exhibit a myopathy with hypotrophic myofibers. Bit-1-null myoblasts prematurely express muscle-specific proteins. Similarly, knockdown of Bit-1 expression in C2C12 myoblasts promotes early differentiation, whereas overexpression delays differentiation. In wild-type mice, Bit-1 levels increase during differentiation. Bit-1-null myoblasts exhibited increased levels of caspase 9 and caspase 3 without increased apoptosis. Bit-1 re-expression partially rescued differentiation. In Bit-1-null muscle, Bcl-2 levels are reduced, suggesting that Bcl-2-mediated inhibition of caspase 9 and caspase 3 is decreased. Bcl-2 re-expression rescued Bit-1-mediated early differentiation in Bit-1-null myoblasts and C2C12 cells with knockdown of Bit-1 expression. These results support an unanticipated yet essential role for Bit-1 in controlling myogenesis through regulation of Bcl-2.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Diferenciación Celular , Desarrollo de Músculos , Animales , Apoptosis , Hidrolasas de Éster Carboxílico/deficiencia , Caspasa 3/metabolismo , Línea Celular , Técnicas de Silenciamiento del Gen , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Fibras Musculares Esqueléticas/patología , Mioblastos/enzimología , Mioblastos/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/metabolismo , TransfecciónRESUMEN
Interleukin (IL) 17-secreting CD4(+) helper T cells (Th17 cells) are essential for host defense at mucosal surfaces, and Th17 cell dysregulation can result in autoimmunity. Exposure to microbial products, such as bacterial LPS, can affect the ability of dendritic cells (DCs) to polarize Th17 cells. Acyloxyacyl hydrolase (AOAH) is a mammalian enzyme expressed by antigen (Ag)-presenting cells that deacylates and thereby inactivates LPS in host tissues. We hypothesized that inactivation of intestinal microbiota-derived LPS by AOAH influences the ability of DCs to polarize and generate Th17 effector cells. We found that LPS-containing Gram-negative microbiota augmented the differentiation of Ag-specific Th17 cells, and identified a colonic DC subset (CD103(+)CD11b(+)ALDH(-)) displaying a unique capacity to both express AOAH and polarize Th17 cells. Compared with WT, these Aoah(-/-) colonic DCs produce less IL-6, resulting in diminished Ag-specific Th17 polarization and increased regulatory T-cell induction in vitro. Oral administration of LPS led to reduced IL-6 production from CD103(+)CD11b(+)ALDH(-) colonic DCs in Aoah(-/-) mice compared with Aoah(+/+) mice, resulting in an abrogated Ag-specific Th17 response in the colon after mucosal immunization that could be rescued by systemic delivery of recombinant IL-6. These data identify the ability of AOAH to modulate microbiota signals that drive Th17 polarization and influence mucosal T-cell immunity, and suggest that host pathways to handle microbiota-derived products may be targeted to modulate Th17 responses in the context of inflammatory disorders or infection at mucosal surfaces.
Asunto(s)
Hidrolasas de Éster Carboxílico/deficiencia , Colon/metabolismo , Células Dendríticas/citología , Mucosa Intestinal/metabolismo , Lipopolisacáridos/metabolismo , Células Th17/citología , Animales , Células Presentadoras de Antígenos/citología , Antígenos CD/metabolismo , Células de la Médula Ósea/citología , Antígeno CD11b/metabolismo , Linfocitos T CD4-Positivos/citología , Hidrolasas de Éster Carboxílico/fisiología , Endotoxinas/metabolismo , Femenino , Citometría de Flujo , Inflamación , Cadenas alfa de Integrinas/metabolismo , Interleucina-6/metabolismo , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteínas Recombinantes/metabolismo , Linfocitos T/citología , Receptores Toll-Like/metabolismoRESUMEN
OBJECTIVE: Liver is the major organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or as bile acids. Intracellular hydrolysis of lipoprotein-derived cholesteryl esters (CEs) is essential to generate the free cholesterol required for this process. Earlier, we demonstrated that overexpression of human CE hydrolase (Gene symbol CES1) increased bile acid synthesis in human hepatocytes and enhanced reverse cholesterol transport in mice. The objective of the present study was to demonstrate that liver-specific deletion of its murine ortholog, Ces3, would decrease cholesterol elimination from the body and increase atherosclerosis. APPROACH AND RESULTS: Liver-specific Ces3 knockout mice (Ces3-LKO) were generated, and Ces3 deficiency did not affect the expression of genes involved in cholesterol homeostasis and free cholesterol or bile acid transport. The effects of Ces3 deficiency on the development of Western diet-induced atherosclerosis were examined in low density lipoprotein receptor knock out(-/-) mice. Despite similar plasma lipoprotein profiles, there was increased lesion development in low density lipoprotein receptor knock out(-/-)Ces3-LKO mice along with a significant decrease in the bile acid content of bile. Ces3 deficiency significantly reduced the flux of cholesterol from [(3)H]-CE-labeled high-density lipoproteins to feces (as free cholesterol and bile acids) and decreased total fecal sterol elimination. CONCLUSIONS: Our results demonstrate that hepatic Ces3 modulates the hydrolysis of lipoprotein-delivered CEs and thereby regulates free cholesterol and bile acid secretion into the feces. Therefore, its deficiency results in reduced cholesterol elimination from the body, leading to significant increase in atherosclerosis. Collectively, these data establish the antiatherogenic role of hepatic CE hydrolysis.
Asunto(s)
Aterosclerosis/genética , Aterosclerosis/metabolismo , Hidrolasas de Éster Carboxílico/genética , Receptores de Lipoproteína/genética , Esteroles/metabolismo , Alimentación Animal , Animales , Ácidos y Sales Biliares/metabolismo , Hidrolasas de Éster Carboxílico/deficiencia , Hidrolasas de Éster Carboxílico/metabolismo , HDL-Colesterol/sangre , LDL-Colesterol/sangre , VLDL-Colesterol/sangre , Heces/enzimología , Femenino , Homeostasis/fisiología , Humanos , Hígado/enzimología , Masculino , Ratones , Ratones Noqueados , Receptores de Lipoproteína/metabolismoRESUMEN
UNLABELLED: Increased lipogenesis, together with hyperlipidemia and increased fat deposition, contribute to obesity and associated metabolic disorders including nonalcoholic fatty liver disease. Here we show that carboxylesterase 1/esterase-x (Ces1/Es-x) plays a regulatory role in hepatic fat metabolism in the mouse. We demonstrate that Ces1/Es-x knockout mice present with increased hepatic lipogenesis and with oversecretion of apolipoprotein B (apoB)-containing lipoproteins (hepatic very-low density lipoproteins), which leads to hyperlipidemia and increased fat deposition in peripheral tissues. Consequently, Ces1/Es-x knockout mice develop obesity, fatty liver, hyperinsulinemia, and insulin insensitivity on chow diet without change in food intake and present with decreased energy expenditure. Ces1/Es-x deficiency prevents the release of polyunsaturated fatty acids from triacylglycerol stores, leading to an up-regulation of sterol regulatory element binding protein 1c-mediated lipogenesis, which can be reversed with dietary ω-3 fatty acids. CONCLUSION: These studies support a role for Ces1/Es-x in the partitioning of regulatory fatty acids and concomitant control of hepatic lipid biosynthesis, secretion, and deposition.
Asunto(s)
Hidrolasas de Éster Carboxílico/deficiencia , Colesterol/metabolismo , Hígado Graso/enzimología , Hiperlipidemias/enzimología , Obesidad/enzimología , Análisis de Varianza , Animales , Glucemia/metabolismo , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Metabolismo Energético , Ayuno , Ácidos Grasos Insaturados/administración & dosificación , Ácidos Grasos Insaturados/metabolismo , Hígado Graso/genética , Femenino , Aceites de Pescado/administración & dosificación , Expresión Génica , Hepatocitos/metabolismo , Hiperlipidemias/genética , Insulina/sangre , Lipoproteínas VLDL/metabolismo , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico , Obesidad/genética , Fenotipo , Fosfolípidos/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/metabolismoRESUMEN
OBJECTIVE: The consequences of macrophage triglyceride (TG) accumulation on atherosclerosis have not been studied in detail so far. Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for the initial step in TG hydrolysis. Because ATGL knockout (KO) mice exhibit massive TG accumulation in macrophages, we used ATGL KO mice to study the effects of macrophage TG accumulation on atherogenesis. METHODS AND RESULTS: Low-density lipoprotein receptor (LDLr) KO mice were transplanted with bone marrow from ATGL KO (ATGL KOâLDLr KO) or wild-type (WTâLDLr KO) mice and challenged with a Western-type diet for 9 weeks. Despite TG accumulation in ATGL KO macrophages, atherosclerosis in ATGL KOâLDLr KO mice was 43% reduced associated with decreased plasma monocyte chemoattractant protein-1 (MCP-1) and macrophage interleukin-6 concentrations. This coincided with a reduced amount of macrophages, possibly because of a 39% increase in intraplaque apoptosis and a decreased migratory capacity of ATGL KO macrophages. The reduced number of white blood cells might be due to a 36% decreased Lin(-)Sca-1(+)cKit(+) hematopoietic stem cell population. CONCLUSIONS: We conclude that the attenuation of atherogenesis in ATGL KOâLDLr KO mice is due to decreased infiltration of less inflammatory macrophages into the arterial wall and increased macrophage apoptosis.
Asunto(s)
Aterosclerosis/prevención & control , Hidrolasas de Éster Carboxílico/deficiencia , Macrófagos/enzimología , Receptores de LDL/deficiencia , Triglicéridos/metabolismo , Animales , Apoptosis , Aterosclerosis/enzimología , Aterosclerosis/etiología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Trasplante de Médula Ósea , Hidrolasas de Éster Carboxílico/genética , Células Cultivadas , Quimiocina CCL2/sangre , Quimiotaxis , Colesterol/sangre , Dieta Aterogénica , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Hidrólisis , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Recuento de Leucocitos , Lipasa , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones , Ratones Noqueados , Células Madre Multipotentes/metabolismo , Receptores de LDL/genética , Triglicéridos/sangre , Irradiación Corporal TotalRESUMEN
There are few, if any, enzymes that have been studied by, and have importance in, so many and varied disciplines as has monocyte esterase/Ces 1. In this review its involvement in the fields of histochemistry, haematology, toxicology, pharmacology, therapeutics, and tumour cell killing, immune surveillance and malignant disease, is briefly described.
Asunto(s)
Hidrolasas de Éster Carboxílico/fisiología , Monocitos/enzimología , Biomarcadores/sangre , Hidrolasas de Éster Carboxílico/deficiencia , Hidrolasas de Éster Carboxílico/genética , Muerte Celular/fisiología , Humanos , Mutación , Neoplasias/enzimología , Neoplasias/patología , Preparaciones Farmacéuticas/metabolismoRESUMEN
In this study, we examined whether ascorbic acid (AA) and dehydroascorbic acid (DHA), the oxidized form of AA, levels in tissues regulate the AA transporters, sodium-dependent vitamin C transporters (SVCT) 1 and SVCT2 and DHA transporters, glucose transporter (GLUT) 1, GLUT3, GLUT4 mRNA by using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are incapable of synthesizing AA in vivo. AA depletion enhanced SVCT1 and SVCT2 mRNA expression in the liver and SVCT1 and GLUT4 mRNA expression in the small intestine, but not in the cerebrum or kidney. Next, we examined the actual impact of AA uptake by using primary cultured hepatocytes from SMP30/GNL KO mice. In the AA-depleted hepatocytes from SMP30/GNL KO mice, AA uptake was significantly greater than in matched cultures from wild-type mice. These results strongly affirm that intracellular AA is an important regulator of SVCT1 and SVCT2 expression in the liver.
Asunto(s)
Ácido Ascórbico/metabolismo , Proteínas de Unión al Calcio/genética , Hidrolasas de Éster Carboxílico/genética , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Hígado/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Simportadores/genética , Animales , Transporte Biológico , Peso Corporal , Proteínas de Unión al Calcio/deficiencia , Hidrolasas de Éster Carboxílico/deficiencia , Ácido Deshidroascórbico/metabolismo , Femenino , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Hígado/citología , Masculino , Ratones , Ratones Noqueados , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transportadores de Sodio Acoplados a la Vitamina C , Simportadores/metabolismoRESUMEN
FAs are mobilized from triglyceride (TG) stores during exercise to supply the working muscle with energy. Mice deficient for adipose triglyceride lipase (ATGL-ko) exhibit defective lipolysis and accumulate TG in adipose tissue and muscle, suggesting that ATGL deficiency affects energy availability and substrate utilization in working muscle. In this study, we investigated the effect of moderate treadmill exercise on blood energy metabolites and liver glycogen stores in mice lacking ATGL. Because ATGL-ko mice exhibit massive accumulation of TG in the heart and cardiomyopathy, we also investigated a mouse model lacking ATGL in all tissues except cardiac muscle (ATGL-ko/CM). In contrast to ATGL-ko mice, these mice did not accumulate TG in the heart and had normal life expectancy. Exercise experiments revealed that ATGL-ko and ATGL-ko/CM mice are unable to increase circulating FA levels during exercise. The reduced availability of FA for energy conversion led to rapid depletion of liver glycogen stores and hypoglycemia. Together, our studies suggest that ATGL-ko mice cannot adjust circulating FA levels to the increased energy requirements of the working muscle, resulting in an increased use of carbohydrates for energy conversion. Thus, ATGL activity is required for proper energy supply of the skeletal muscle during exercise.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Ácidos Grasos/metabolismo , Músculos/metabolismo , Animales , Carbohidratos/sangre , Hidrolasas de Éster Carboxílico/deficiencia , Hidrolasas de Éster Carboxílico/genética , Metabolismo Energético , Femenino , Técnicas de Inactivación de Genes , Glucógeno/metabolismo , Lipasa , Lípidos/sangre , Hígado/metabolismo , Locomoción , Masculino , Ratones , Músculos/citología , Músculos/fisiología , Mutación , Condicionamiento Físico Animal , DescansoRESUMEN
Senescence marker protein-30 (SMP30) is an androgen-independent factor that decreases with age. We recently identified SMP30 as the lactone-hydrolyzing enzyme gluconolactonase (GNL), which is involved in vitamin C biosynthesis in animal species. To examine whether the age-related decrease in SMP30/GNL has effects on glucose homeostasis, we used SMP30/GNL knockout (KO) mice treated with L-ascorbic acid. In an ip glucose tolerance test at 15 wk of age, blood glucose levels in SMP30/GNL KO mice were significantly increased by 25% at 30 min after glucose administration compared with wild-type (WT) mice. Insulin levels in SMP30/GNL KO mice were significantly decreased by 37% at 30 min after glucose compared with WT mice. Interestingly, an insulin tolerance test showed a greater glucose-lowering effect in SMP30/GNL KO mice. High-fat diet feeding severely worsened glucose tolerance in both WT and SMP30/GNL KO mice. Morphometric analysis revealed no differences in the degree of high-fat diet-induced compensatory increase in beta-cell mass and proliferation. In the static incubation study of islets, insulin secretion in response to 20 mm glucose or KCl was significantly decreased in SMP30/GNL KO mice. On the other hand, islet ATP content at 20 mm in SMP30/GNL KO mice was similar to that in WT mice. Collectively, these data indicate that impairment of the early phase of insulin secretion due to dysfunction of the distal portion of the secretion pathway underlies glucose intolerance in SMP30/GNL KO mice. Decreased SMP30/GNL may contribute to the worsening of glucose tolerance that occurs in normal aging.
Asunto(s)
Proteínas de Unión al Calcio/deficiencia , Hidrolasas de Éster Carboxílico/deficiencia , Hidrolasas de Éster Carboxílico/genética , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/fisiología , Envejecimiento/genética , Envejecimiento/fisiología , Alimentación Animal , Animales , Apoptosis/efectos de los fármacos , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Encéfalo/fisiología , División Celular/efectos de los fármacos , Epidídimo/anatomía & histología , Prueba de Tolerancia a la Glucosa , Insulina/análisis , Insulina/farmacología , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/patología , Islotes Pancreáticos/fisiología , Hígado/citología , Hígado/efectos de los fármacos , Hígado/fisiología , Pulmón/fisiología , Masculino , Ratones , Ratones Noqueados , Alveolos Pulmonares/patología , Factor de Necrosis Tumoral alfa/farmacología , Aumento de Peso , Receptor fas/farmacologíaRESUMEN
The enzyme neuropathy target esterase (NTE) is present in neurons and deacylates the major membrane phospholipid, phosphatidylcholine (PtdCho). Mutation of the NTE gene or poisoning by neuropathic organophosphates--chemical inhibitors of NTE--causes distal degeneration of long spinal axons in humans. However, analogous neuropathological changes have not been reported in nestin-cre:NTEfl/fl mice with NTE-deficient neural tissue. Furthermore, altered PtdCho homeostasis has not been detected in NTE-deficient vertebrates. Here, we describe distal degeneration of the longest spinal axons in approximately 3-week-old nestin-cre:NTEfl/fl mice and in adult C57BL/6J mice after acute dosing with a neuropathic organophosphate: in both groups early degenerative lesions were followed by swellings comprising accumulated axoplasmic material. In mice dosed acutely with organophosphate, maximal numbers of lesions, in the longest spinal sensory axon tract, were attained within days and were preceded by a transient rise in neural PtdCho. In nestin-cre:NTEfl/fl mice, sustained elevation of PtdCho over many months was accompanied by progressive degeneration and massive swelling of axons in sensory and motor spinal tracts and by increasing hindlimb dysfunction. Axonal lesion distribution closely resembled that in hereditary spastic paraplegia (HSP). The importance of defective membrane trafficking in HSP and the association of NTE with the endoplasmic reticulum--the starting point for the constitutive secretory pathway and transport of neuronal materials into axons--prompted investigation for a role of NTE in secretion. Cultured NTE-deficient neurons displayed modestly impaired secretion, consistent with neuronal viability and damage in vivo initially restricted to distal parts of the longest axons.
Asunto(s)
Axones/fisiología , Hidrolasas de Éster Carboxílico/metabolismo , Enfermedades del Sistema Nervioso/patología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Axones/enzimología , Axones/ultraestructura , Hidrolasas de Éster Carboxílico/deficiencia , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/patología , Proteínas de Filamentos Intermediarios/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Degeneración Nerviosa/etiología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/genética , Enfermedades del Sistema Nervioso/inducido químicamente , Enfermedades del Sistema Nervioso/enzimología , Enfermedades del Sistema Nervioso/fisiopatología , Nestina , Neuronas/metabolismo , Compuestos Organofosforados , Fosfatidilcolinas/metabolismo , Tractos Piramidales/patología , Médula Espinal/patologíaRESUMEN
We consider here a previously neglected aspect of recovery from infectious diseases: how animals dispose of the dead microbes in their tissues. For one of the most important disease-causing microorganisms, Gram-negative bacteria, there is now evidence that the host catabolism of a key microbial molecule is essential for full recovery. As might be expected, it is the same bacterial molecule that animals sense to detect the presence of Gram-negative bacteria in their tissues, the cell wall lipopolysaccharide (LPS). Here, we discuss current knowledge about LPS sensing with emphasis on the host enzyme that inactivates this microbial "messenger" molecule. We also consider the possibility that the rate at which stimulatory microbial molecules undergo inactivation may influence the duration and severity of diseases caused by other infectious agents.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Bacterias Gramnegativas/citología , Bacterias Gramnegativas/metabolismo , Lipopolisacáridos/metabolismo , Animales , Antibacterianos/uso terapéutico , Hidrolasas de Éster Carboxílico/deficiencia , Hidrolasas de Éster Carboxílico/genética , Bacterias Gramnegativas/química , Bacterias Gramnegativas/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/metabolismo , Humanos , Lipopolisacáridos/química , Viabilidad Microbiana/efectos de los fármacosRESUMEN
BACKGROUND: It has been recently reported that serum paraoxonase (PON1) and arylesterase (ARE) activities may be significantly reduced in patients with chronic liver disease. The aim of the study was to investigate the relations between serum PON1 and ARE activities and the degree of liver damage in patients with chronic liver injury. METHODS: We studied a total of 75 patients with chronic liver disease (50 patients with cirrhosis and 25 patients with chronic hepatitis) and 25 healthy comparison subjects. Baseline and salt-stimulated PON1 and ARE activities were determined in all study participants. RESULTS: Baseline and stimulated PON1 and ARE activities were significantly lower in patients with chronic liver disease than in controls. Cirrhotic patients in Child-Pugh classes B and C subgroups had significantly reduced PON1 and ARE activities compared with Child-Pugh class A patients (both P-values <0.01). Receiver operating characteristic curve analysis showed that serum ARE activity was the most efficient test for identifying the presence and severity of chronic liver injury. CONCLUSION: Baseline and stimulated PON1 and ARE activities are reduced in patients with chronic liver disease. Serum ARE activity could be a suitable biomarker for the evaluation of the presence and severity of chronic liver damage.
Asunto(s)
Arildialquilfosfatasa/sangre , Hidrolasas de Éster Carboxílico/sangre , Hepatitis B Crónica/enzimología , Hepatitis C Crónica/enzimología , Cirrosis Hepática/enzimología , Adulto , Anciano , Alanina Transaminasa/sangre , Apolipoproteína A-I/sangre , Arildialquilfosfatasa/deficiencia , Aspartato Aminotransferasas/sangre , Bilirrubina/sangre , Biomarcadores , Hidrolasas de Éster Carboxílico/deficiencia , Colesterol/sangre , Femenino , Hepatitis B Crónica/sangre , Hepatitis C Crónica/sangre , Humanos , Cirrosis Hepática/sangre , Masculino , Persona de Mediana Edad , Fenotipo , Curva ROC , Índice de Severidad de la Enfermedad , Cloruro de Sodio/farmacología , Estimulación Química , Triglicéridos/sangreRESUMEN
Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous beta-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL(-/-) mice indicated the presence of other TG lipase(s) in the beta-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The K(ATP)-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL(-/-) mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL(-/-) mice. Accordingly, isolated islets from ATGL(-/-) mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL(-/-) islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion.