RESUMO
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.
Assuntos
Comportamento Animal , Hidrolases de Éster Carboxílico/deficiência , Enzimas Reparadoras do DNA/deficiência , Hidrolases/deficiência , Animais , Peso Corporal , Hidrolases de Éster Carboxílico/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Feminino , Marcha , Técnicas de Inativação de Genes , Genótipo , Hidrolases/metabolismo , Masculino , Camundongos Knockout , Atividade Motora , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Neurônios/enzimologia , Bexiga Urinária/inervação , Transtornos Urinários/enzimologia , Micção , Urodinâmica , Animais , Compostos Azo/farmacologia , Núcleo de Barrington/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hidrolases de Éster Carboxílico/deficiência , Hidrolases de Éster Carboxílico/genética , Hormônio Liberador da Corticotropina/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Contração Muscular , Neurônios/efeitos dos fármacos , PPAR gama/genética , PPAR gama/metabolismo , Pressão , Pirazóis/farmacologia , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Bexiga Urinária/efeitos dos fármacos , Micção/efeitos dos fármacos , Transtornos Urinários/tratamento farmacológico , Transtornos Urinários/genética , Transtornos Urinários/fisiopatologia , Urodinâmica/efeitos dos fármacosRESUMO
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.
Assuntos
Controle Biológico de Vetores , Pseudomonas chlororaphis/genética , Percepção de Quorum/genética , Regulon/genética , Proteínas de Bactérias/genética , Hidrolases de Éster Carboxílico/deficiência , Movimento Celular/genética , Quitinases/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Mutantes , RNA-Seq , Sideróforos/genética , Transativadores/genética , TranscriptomaRESUMO
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.
Assuntos
Azotobacter vinelandii/enzimologia , Azotobacter vinelandii/metabolismo , Hidrolases de Éster Carboxílico/deficiência , Hidroxibutiratos/química , Hidroxibutiratos/metabolismo , Poliésteres/química , Poliésteres/metabolismo , Reatores Biológicos/microbiologia , Hidrolases de Éster Carboxílico/metabolismo , Deleção de Genes , Peso MolecularRESUMO
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.
Assuntos
Hidrolases de Éster Carboxílico/deficiência , Mutação/genética , Sítios de Splice de RNA/genética , Paraplegia Espástica Hereditária/genética , Sequência de Bases , Biomarcadores/metabolismo , Líquidos Corporais/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Família , Feminino , Fibroblastos/metabolismo , Genômica , Homozigoto , Humanos , Íntrons/genética , Imageamento por Ressonância Magnética , Masculino , Linhagem , Fenótipo , Fosfatidilgliceróis/metabolismoRESUMO
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.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Cistite Intersticial/enzimologia , Infecções por Escherichia coli/enzimologia , Hiperalgesia/enzimologia , Dor Pélvica/enzimologia , Pseudorraiva/enzimologia , Bexiga Urinária/inervação , Infecções Urinárias/enzimologia , Animais , Comportamento Animal , Hidrolases de Éster Carboxílico/deficiência , Hidrolases de Éster Carboxílico/genética , Cistite Intersticial/genética , Cistite Intersticial/fisiopatologia , Cistite Intersticial/psicologia , Modelos Animais de Doenças , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/fisiopatologia , Infecções por Escherichia coli/psicologia , Feminino , Predisposição Genética para Doença , Hiperalgesia/genética , Hiperalgesia/fisiopatologia , Hiperalgesia/psicologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Percepção da Dor , Limiar da Dor , Dor Pélvica/genética , Dor Pélvica/fisiopatologia , Fenótipo , Pseudorraiva/genética , Pseudorraiva/fisiopatologia , Pseudorraiva/psicologia , Locos de Características Quantitativas , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/metabolismo , Bexiga Urinária/metabolismo , Infecções Urinárias/genética , Infecções Urinárias/fisiopatologia , Infecções Urinárias/psicologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Fígado Gorduroso/prevenção & controle , Terapia Genética , Hiperlipidemias/prevenção & controle , Resistência à Insulina , Insulina/sangue , Metabolismo dos Lipídeos , Fígado/enzimologia , Animais , Glicemia/metabolismo , Hidrolases de Éster Carboxílico/deficiência , Hidrolases de Éster Carboxílico/genética , Modelos Animais de Doenças , Fígado Gorduroso/sangue , Fígado Gorduroso/enzimologia , Fígado Gorduroso/genética , Feminino , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Predisposição Genética para Doença , Hiperlipidemias/sangue , Hiperlipidemias/enzimologia , Hiperlipidemias/genética , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/genética , Lipoproteínas VLDL/sangue , Camundongos Knockout , Fenótipo , Transdução de Sinais , Fatores de Tempo , Triglicerídeos/sangueRESUMO
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.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Diferenciação Celular , Desenvolvimento Muscular , Animais , Apoptose , Hidrolases de Éster Carboxílico/deficiência , Caspase 3/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fibras Musculares Esqueléticas/patologia , Mioblastos/enzimologia , Mioblastos/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Hidrolases de Éster Carboxílico/deficiência , Colo/metabolismo , Células Dendríticas/citologia , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/metabolismo , Células Th17/citologia , Animais , Células Apresentadoras de Antígenos/citologia , Antígenos CD/metabolismo , Células da Medula Óssea/citologia , Antígeno CD11b/metabolismo , Linfócitos T CD4-Positivos/citologia , Hidrolases de Éster Carboxílico/fisiologia , Endotoxinas/metabolismo , Feminino , Citometria de Fluxo , Inflamação , Cadeias alfa de Integrinas/metabolismo , Interleucina-6/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteínas Recombinantes/metabolismo , Linfócitos T/citologia , Receptores Toll-Like/metabolismoRESUMO
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.
Assuntos
Aterosclerose/genética , Aterosclerose/metabolismo , Hidrolases de Éster Carboxílico/genética , Receptores de Lipoproteínas/genética , Esteróis/metabolismo , Ração Animal , Animais , Ácidos e Sais Biliares/metabolismo , Hidrolases de Éster Carboxílico/deficiência , Hidrolases de Éster Carboxílico/metabolismo , HDL-Colesterol/sangue , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Fezes/enzimologia , Feminino , Homeostase/fisiologia , Humanos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Knockout , Receptores de Lipoproteínas/metabolismoRESUMO
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.
Assuntos
Hidrolases de Éster Carboxílico/deficiência , Colesterol/metabolismo , Fígado Gorduroso/enzimologia , Hiperlipidemias/enzimologia , Obesidade/enzimologia , Análise de Variância , Animais , Glicemia/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Metabolismo Energético , Jejum , Ácidos Graxos Insaturados/administração & dosagem , Ácidos Graxos Insaturados/metabolismo , Fígado Gorduroso/genética , Feminino , Óleos de Peixe/administração & dosagem , Expressão Gênica , Hepatócitos/metabolismo , Hiperlipidemias/genética , Insulina/sangue , Lipoproteínas VLDL/metabolismo , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica , Obesidade/genética , Fenótipo , Fosfolipídeos/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/metabolismoRESUMO
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.
Assuntos
Hidrolases de Éster Carboxílico/fisiologia , Monócitos/enzimologia , Biomarcadores/sangue , Hidrolases de Éster Carboxílico/deficiência , Hidrolases de Éster Carboxílico/genética , Morte Celular/fisiologia , Humanos , Mutação , Neoplasias/enzimologia , Neoplasias/patologia , Preparações Farmacêuticas/metabolismoRESUMO
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.
Assuntos
Aterosclerose/prevenção & controle , Hidrolases de Éster Carboxílico/deficiência , Macrófagos/enzimologia , Receptores de LDL/deficiência , Triglicerídeos/metabolismo , Animais , Apoptose , Aterosclerose/enzimologia , Aterosclerose/etiologia , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Transplante de Medula Óssea , Hidrolases de Éster Carboxílico/genética , Células Cultivadas , Quimiocina CCL2/sangue , Quimiotaxia , Colesterol/sangue , Dieta Aterogênica , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Hidrólise , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Contagem de Leucócitos , Lipase , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Células-Tronco Multipotentes/metabolismo , Receptores de LDL/genética , Triglicerídeos/sangue , Irradiação Corporal TotalRESUMO
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.
Assuntos
Ácido Ascórbico/metabolismo , Proteínas de Ligação ao Cálcio/genética , Hidrolases de Éster Carboxílico/genética , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fígado/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética , Animais , Transporte Biológico , Peso Corporal , Proteínas de Ligação ao Cálcio/deficiência , Hidrolases de Éster Carboxílico/deficiência , Ácido Desidroascórbico/metabolismo , Feminino , Proteínas Facilitadoras de Transporte de Glucose/genética , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Fígado/citologia , Masculino , Camundongos , Camundongos Knockout , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transportadores de Sódio Acoplados à Vitamina C , Simportadores/metabolismoRESUMO
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.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Ácidos Graxos/metabolismo , Músculos/metabolismo , Animais , Carboidratos/sangue , Hidrolases de Éster Carboxílico/deficiência , Hidrolases de Éster Carboxílico/genética , Metabolismo Energético , Feminino , Técnicas de Inativação de Genes , Glicogênio/metabolismo , Lipase , Lipídeos/sangue , Fígado/metabolismo , Locomoção , Masculino , Camundongos , Músculos/citologia , Músculos/fisiologia , Mutação , Condicionamento Físico Animal , DescansoRESUMO
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.
Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Hidrolases de Éster Carboxílico/deficiência , Hidrolases de Éster Carboxílico/genética , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/fisiologia , Envelhecimento/genética , Envelhecimento/fisiologia , Ração Animal , Animais , Apoptose/efeitos dos fármacos , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Encéfalo/fisiologia , Divisão Celular/efeitos dos fármacos , Epididimo/anatomia & histologia , Teste de Tolerância a Glucose , Insulina/análise , Insulina/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/fisiologia , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/fisiologia , Pulmão/fisiologia , Masculino , Camundongos , Camundongos Knockout , Alvéolos Pulmonares/patologia , Fator de Necrose Tumoral alfa/farmacologia , Aumento de Peso , Receptor fas/farmacologiaRESUMO
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.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Bactérias Gram-Negativas/citologia , Bactérias Gram-Negativas/metabolismo , Lipopolissacarídeos/metabolismo , Animais , Antibacterianos/uso terapêutico , Hidrolases de Éster Carboxílico/deficiência , Hidrolases de Éster Carboxílico/genética , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/metabolismo , Humanos , Lipopolissacarídeos/química , Viabilidade Microbiana/efeitos dos fármacosRESUMO
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.
Assuntos
Axônios/fisiologia , Hidrolases de Éster Carboxílico/metabolismo , Doenças do Sistema Nervoso/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Axônios/enzimologia , Axônios/ultraestrutura , Hidrolases de Éster Carboxílico/deficiência , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Retículo Endoplasmático/patologia , Proteínas de Filamentos Intermediários/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Degeneração Neural/etiologia , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/genética , Doenças do Sistema Nervoso/induzido quimicamente , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/fisiopatologia , Nestina , Neurônios/metabolismo , Compostos Organofosforados , Fosfatidilcolinas/metabolismo , Tratos Piramidais/patologia , Medula Espinal/patologiaRESUMO
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.
Assuntos
Arildialquilfosfatase/sangue , Hidrolases de Éster Carboxílico/sangue , Hepatite B Crônica/enzimologia , Hepatite C Crônica/enzimologia , Cirrose Hepática/enzimologia , Adulto , Idoso , Alanina Transaminase/sangue , Apolipoproteína A-I/sangue , Arildialquilfosfatase/deficiência , Aspartato Aminotransferases/sangue , Bilirrubina/sangue , Biomarcadores , Hidrolases de Éster Carboxílico/deficiência , Colesterol/sangue , Feminino , Hepatite B Crônica/sangue , Hepatite C Crônica/sangue , Humanos , Cirrose Hepática/sangue , Masculino , Pessoa de Meia-Idade , Fenótipo , Curva ROC , Índice de Gravidade de Doença , Cloreto de Sódio/farmacologia , Estimulação Química , Triglicerídeos/sangueRESUMO
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.