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Ceramides and cardiorespiratory (CR) fitness are both related to cardiovascular diseases. The associations of three blood plasma ceramides (C16:0, C22:0, and C24:0) with CR fitness in the population-based Study of Health in Pomerania (SHIP-START-1; n = 1,102; mean age 50.3 years, 51.5% women) are investigated. In addition, subgroup analysis according to age (≥54 years) and sex (female/male) is performed. Ceramides are quantified by liquid chromatography/mass spectrometry (LC/MS). CR fitness is assessed by a cardiopulmonary exercise test. Sex and age independent associations are found for higher levels of C24:0 and C24:0/C16:0 ratio with higher maximal oxygen consumption (VO2peak) kg-1 and oxygen consumption at the anaerobic threshold (VO2@AT1) as well as for the relation of C24:0/C16:0 with maximum workload (Wattmax kg-1). In contrast, age/sex subgroup specific inverse associations with Wattmax kg-1 are found in women <54 years for C22:0, while a positive association in men ≥54 years. Higher levels of C24:0 are associated with higher Wattmax kg-1, except for women <54 years, where no significant association can be found. The findings suggest that the use of single ceramides as cardiovascular biomarkers may be inferior, compared to ceramide ratio C24:0/C16:0. Therefore C24:0/C16:0 ratio may be a more suitable and robust cardiovascular biomarker and should be preferred over single ceramides.
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Capacidad Cardiovascular , Enfermedades Cardiovasculares , Humanos , Masculino , Femenino , Persona de Mediana Edad , Ceramidas , Biomarcadores , Enfermedades Cardiovasculares/epidemiologíaRESUMEN
Pancreatic ß cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair ß cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion is similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on ß cell mRNA translation. Before induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during ß cell glucose toxicity that impairs expression of proteins with critical roles in ß cell function.
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Hiperglucemia , Células Secretoras de Insulina , Islotes Pancreáticos , Neoplasias Pancreáticas , Ratas , Humanos , Animales , Secreción de Insulina , ARN Mensajero/metabolismo , Insulina/metabolismo , Hiperglucemia/genética , Hiperglucemia/metabolismo , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Péptidos/metabolismo , Neoplasias Pancreáticas/metabolismo , Islotes Pancreáticos/metabolismoRESUMEN
Pancreatic ß-cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair ß-cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion are similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on ß-cell mRNA translation. Prior to induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also of mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex-vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our findings uncover a translational regulatory circuit during ß-cell glucose toxicity that impairs expression of proteins with critical roles in ß-cell function.
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BACKGROUND: GM1 gangliosidosis is a rare, fatal, neurodegenerative disease caused by mutations in the GLB1 gene and deficiency in ß-galactosidase. Delay of symptom onset and increase in lifespan in a GM1 gangliosidosis cat model after adeno-associated viral (AAV) gene therapy treatment provide the basis for AAV gene therapy trials. The availability of validated biomarkers would greatly improve assessment of therapeutic efficacy. METHODS: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to screen oligosaccharides as potential biomarkers for GM1 gangliosidosis. The structures of pentasaccharide biomarkers were determined with mass spectrometry, as well as chemical and enzymatic degradations. Comparison of LC-MS/MS data of endogenous and synthetic compounds confirmed the identification. The study samples were analyzed with fully validated LC-MS/MS methods. FINDINGS: We identified two pentasaccharide biomarkers, H3N2a and H3N2b, that were elevated more than 18-fold in patient plasma, cerebrospinal fluid (CSF), and urine. Only H3N2b was detectable in the cat model, and it was negatively correlated with ß-galactosidase activity. Following intravenous (IV) AAV9 gene therapy treatment, reduction of H3N2b was observed in central nervous system, urine, plasma, and CSF samples from the cat model and in urine, plasma, and CSF samples from a patient. Reduction of H3N2b accurately reflected normalization of neuropathology in the cat model and improvement of clinical outcomes in the patient. INTERPRETATIONS: These results demonstrate that H3N2b is a useful pharmacodynamic biomarker to evaluate the efficacy of gene therapy for GM1 gangliosidosis. H3N2b will facilitate the translation of gene therapy from animal models to patients. FUNDING: This work was supported by grants U01NS114156, R01HD060576, ZIAHG200409, and P30 DK020579 from the National Institutes of Health (NIH) and a grant from National Tay-Sachs and Allied Diseases Association Inc.
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Gangliosidosis GM1 , Enfermedades Neurodegenerativas , Animales , Gangliosidosis GM1/genética , Gangliosidosis GM1/terapia , Gangliosidosis GM1/patología , Enfermedades Neurodegenerativas/terapia , Cromatografía Liquida , Espectrometría de Masas en Tándem , beta-Galactosidasa/genética , beta-Galactosidasa/química , beta-Galactosidasa/uso terapéutico , Biomarcadores/líquido cefalorraquídeo , Terapia GenéticaRESUMEN
Dyslipidemia and resulting lipotoxicity are pathologic signatures of metabolic syndrome and type 2 diabetes. Excess lipid causes cell dysfunction and induces cell death through pleiotropic mechanisms that link to oxidative stress. However, pathways that regulate the response to metabolic stress are not well understood. Herein, we show that disruption of the box H/ACA SNORA73 small nucleolar RNAs encoded within the small nucleolar RNA hosting gene 3 (Snhg3) causes resistance to lipid-induced cell death and general oxidative stress in cultured cells. This protection from metabolic stress is associated with broad reprogramming of oxidative metabolism that is dependent on the mammalian target of rapamycin signaling axis. Furthermore, we show that knockdown of SNORA73 in vivo protects against hepatic steatosis and lipid-induced oxidative stress and inflammation. Our findings demonstrate a role for SNORA73 in the regulation of metabolism and lipotoxicity.
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Hígado Graso/tratamiento farmacológico , Hígado Graso/metabolismo , Sustancias Protectoras/farmacología , ARN Nucleolar Pequeño/metabolismo , Animales , Células CHO , Muerte Celular/efectos de los fármacos , Cricetulus , Diabetes Mellitus Tipo 2/metabolismo , Hígado Graso/genética , Homeostasis , Inflamación , Metabolismo de los Lípidos , Lípidos/farmacología , Masculino , Síndrome Metabólico/metabolismo , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , ARN Largo no Codificante , ARN Nucleolar Pequeño/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.
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Colesterol/análogos & derivados , Diazometano/análogos & derivados , Canales Iónicos Activados por Ligandos/química , Etiquetas de Fotoafinidad/química , Alquinos/síntesis química , Alquinos/química , Alquinos/metabolismo , Sitios de Unión , Colesterol/síntesis química , Colesterol/metabolismo , Cianobacterias/química , Diazometano/síntesis química , Diazometano/metabolismo , Colorantes Fluorescentes/química , Canales Iónicos Activados por Ligandos/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Etiquetas de Fotoafinidad/síntesis química , Etiquetas de Fotoafinidad/metabolismo , Unión ProteicaRESUMEN
Niemann-Pick disease type C (NPC) is a neurodegenerative disease in which mutation of NPC1 or NPC2 gene leads to lysosomal accumulation of unesterified cholesterol and sphingolipids. Diagnosis of NPC disease is challenging due to non-specific early symptoms. Biomarker and genetic tests are used as first-line diagnostic tests for NPC. In this study, we developed a plasma test based on N-(3ß,5α,6ß-trihydroxy-cholan-24-oyl)glycine (TCG) that was markedly increased in the plasma of human NPC1 subjects. The test showed sensitivity of 0.9945 and specificity of 0.9982 to differentiate individuals with NPC1 from NPC1 carriers and controls. Compared to other commonly used biomarkers, cholestane-3ß,5α,6ß-triol (C-triol) and N-palmitoyl-O-phosphocholine (PPCS, also referred to as lysoSM-509), TCG was equally sensitive for identifying NPC1 but more specific. Unlike C-triol and PPCS, TCG showed excellent stability and no spurious generation of marker in the sample preparation or aging of samples. TCG was also elevated in lysosomal acid lipase deficiency (LALD) and acid sphingomyelinase deficiency (ASMD). Plasma TCG was significantly reduced after intravenous (IV) 2-hydroxypropyl-ß-cyclodextrin (HPßCD) treatment. These results demonstrate that plasma TCG was superior to C-triol and PPCS as NPC1 diagnostic biomarker and was able to evaluate the peripheral treatment efficacy of IV HPßCD treatment.
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Glicina/sangre , Péptidos y Proteínas de Señalización Intracelular/genética , Enfermedad de Niemann-Pick Tipo C/sangre , Enfermedad de Niemann-Pick Tipo C/genética , 2-Hidroxipropil-beta-Ciclodextrina/administración & dosificación , Ácidos y Sales Biliares/sangre , Biomarcadores/sangre , Femenino , Glicina/análogos & derivados , Glicina/aislamiento & purificación , Humanos , Masculino , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Enfermedad de Niemann-Pick Tipo C/patología , Espectrometría de Masas en Tándem , Proteínas de Transporte Vesicular/genéticaRESUMEN
OBJECTIVE: Total ceramide concentrations are linked with increased insulin resistance and cardiac dysfunction. However, recent studies have demonstrated that plasma concentrations of specific very-long-chain fatty ceramides (C24:0 and C22:0) are associated with a reduced incidence of coronary heart disease and all-cause mortality. We hypothesized that specific genetic loci are associated with plasma C22:0 and C24:0 concentrations. METHODS: Heritability and genome-wide association studies of plasma C24:0 and C22:0 ceramide concentrations were performed among 2,217 participants in the Framingham Heart Study Offspring Cohort, adjusting for cardiovascular risk factor covariates and cardiovascular drug treatment. RESULTS: The multivariable-adjusted heritability for C22:0 and C24:0 ceramides was 0.42 (standard error [SE], 0.07; p=1.8E-9) and 0.25 (SE, 0.08; p=0.00025), respectively. Nineteen single nucleotide polymorphisms (SNPs), all on chromosome 20, significantly associated with C22:0 concentrations; the closest gene to these variants was SPTLC3. The lead SNP (rs4814175) significantly associated with 3% lower plasma C22:0 concentrations (p=2.83E-11). Nine SNPs, all on chromosome 20 and close to SPTLC3, were significantly associated with C24:0 ceramide concentrations. All 9 were also significantly related to plasma C22:0 levels. The lead SNP (rs168622) was significantly associated with 10% lower plasma C24:0 ceramide concentrations (p=9.94E-09). CONCLUSION: SNPs near the SPTLC3 gene, which encodes serine palmitoyltransferase long chain base subunit 3 (SPTLC3; part of the enzyme that catalyzes the rate-limiting step of de novo sphingolipid synthesis) were associated with plasma C22:0 and C24:0 ceramide concentrations. These results are biologically plausible and suggest that SPTLC3 may be a potential therapeutic target for C24:0 and C22:0 ceramide modulation.
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Cardiac dysfunction in T2D is associated with excessive FA uptake, oxidation, and generation of toxic lipid species by the heart. It is not known whether decreasing lipid delivery to the heart can effect improvement in cardiac function in humans with T2D. Thus, our objective was to test the hypothesis that lowering lipid delivery to the heart would result in evidence of decreased "lipotoxicity," improved cardiac function, and salutary effects on plasma biomarkers of cardiovascular risk. Thus, we performed a double-blind randomized placebo-controlled parallel design study of the effects of 12 weeks of fenofibrate-induced lipid lowering on cardiac function, inflammation, and oxidation biomarkers, and on the ratio of two plasma ceramides, Cer d18:1 (4E) (1OH, 3OH)/24:0 and Cer d18:1 (4E) (1OH, 3OH)/16:0 (i.e., "C24:0/C16:0"), which is associated with decreased risk of cardiac dysfunction and heart failure. Fenofibrate lowered plasma TG and cholesterol but did not improve heart systolic or diastolic function. Fenofibrate treatment lowered the plasma C24:0/C16:0 ceramide ratio and minimally altered oxidative stress markers but did not alter measures of inflammation. Overall, plasma TG lowering correlated with improvement of cardiac relaxation (diastolic function) as measured by tissue Doppler-derived parameter e'. Moreover, lowering the plasma C24:0/C16:0 ceramide ratio was correlated with worse diastolic function. These findings indicate that fenofibrate treatment per se is not sufficient to effect changes in cardiac function; however, decreases in plasma TG may be linked to improved diastolic function. In contrast, decreases in plasma C24:0/C16:0 are linked with worsening cardiac function.
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Ceramidas/sangre , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/fisiopatología , Fenofibrato/uso terapéutico , Corazón/efectos de los fármacos , Corazón/fisiopatología , Triglicéridos/sangre , Adulto , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Excess fatty acid accumulation in nonadipose tissues leads to cell dysfunction and cell death that is linked to the pathogenesis of inherited and acquired human diseases. Study of this process, known as lipotoxicity, has provided new insights into the regulation of lipid homeostasis and has revealed new molecular pathways involved in lipid-induced cellular stress. The discovery that disruption of specific small nucleolar RNAs protects against fatty acid-induced cell death and remodels metabolism in vivo opens new opportunities for understanding how nutrient signals influence cellular and systemic metabolic homeostasis through RNA biology.
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Muerte Celular , Ácidos Grasos/metabolismo , ARN Nucleolar Pequeño/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ácidos Grasos/toxicidad , Humanos , Síndrome Metabólico/genética , Síndrome Metabólico/metabolismo , Síndrome Metabólico/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Niemann-Pick type C (NPC) disease is a rare lysosomal storage disorder caused by mutations in either the NPC1 or the NPC2 gene. A new class of lipids, N-acyl-O-phosphocholineserines were recently identified as NPC biomarkers. The most abundant species in this class of lipid, N-palmitoyl-O-phosphocholineserine (PPCS), was evaluated for diagnosis of NPC disease and treatment efficacy assessment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) in NPC. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated to measure PPCS in human plasma and cerebrospinal fluid (CSF). A cutoff of 248 ng/mL in plasma provided a sensitivity of 100.0% and specificity of 96.6% in identifying NPC1 patients from control and NPC1 carrier subjects. PPCS was significantly elevated in CSF from NPC1 patients, and CSF PPCS levels were significantly correlated with NPC neurological disease severity scores. Plasma and CSF PPCS did not change significantly in response to intrathetical (IT) HPßCD treatment. In an intravenous (IV) HPßCD trial, plasma PPCS in all patients was significantly reduced. These results demonstrate that plasma PPCS was able to diagnose NPC1 patients with high sensitivity and specificity, and to evaluate the peripheral treatment efficacy of IV HPßCD treatment.
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2-Hidroxipropil-beta-Ciclodextrina/uso terapéutico , Enfermedad de Niemann-Pick Tipo C/diagnóstico , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Fosforilcolina/sangre , Fosforilcolina/líquido cefalorraquídeo , Adolescente , Adulto , Anciano , Animales , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Gatos , Niño , Preescolar , Cromatografía Liquida , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad , Espectrometría de Masas en Tándem , Resultado del Tratamiento , Adulto JovenRESUMEN
Niemann-Pick disease type C (NPC) disease is a lipid-storage disorder that is caused by mutations in the genes encoding NPC proteins and results in lysosomal cholesterol accumulation. 2-Hydroxypropyl-ß-cyclodextrin (CD) has been shown to reduce lysosomal cholesterol levels and enhance sterol homeostatic responses, but CD's mechanism of action remains unknown. Recent work provides evidence that CD stimulates lysosomal exocytosis, raising the possibility that lysosomal cholesterol is released in exosomes. However, therapeutic concentrations of CD do not alter total cellular cholesterol, and cholesterol homeostatic responses at the ER are most consistent with increased ER membrane cholesterol. To address these disparate findings, here we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient U2OS cells. Although released cholesterol was detectable, it was not associated with extracellular vesicles. Rather, we demonstrate that lysosomal cholesterol trafficks to the plasma membrane (PM), where it exchanges with lipoprotein-bound cholesterol in a CD-dependent manner. We found that in the absence of suitable extracellular cholesterol acceptors, cholesterol exchange is abrogated, cholesterol accumulates in the PM, and reesterification at the ER is increased. These results support a model in which CD promotes intracellular redistribution of lysosomal cholesterol, but not cholesterol exocytosis or efflux, during the restoration of cholesterol homeostatic responses.
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Colesterol/metabolismo , Ciclodextrinas/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colesterol/análisis , Homeostasis/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Marcaje Isotópico , Lisosomas/química , Lisosomas/metabolismo , Proteína Niemann-Pick C1RESUMEN
BACKGROUND: We determined the association between ratios of plasma ceramide species of differing fatty-acyl chain lengths and incident dementia and Alzheimer's disease (AD) dementia in a large, community-based sample. METHODS: We measured plasma ceramide levels in 1892 [54% women, mean age 70.1 (SD 6.9) yr.] dementia-free Framingham Offspring Study cohort participants between 2005 and 2008. We related ratios of very long-chain (C24:0, C22:0) to long-chain (C16:0) ceramides to subsequent risk of incident dementia and AD dementia. Structural MRI brain measures were included as secondary outcomes. RESULTS: During a median 6.5 year follow-up, 81 participants developed dementia, of whom 60 were diagnosed with AD dementia. In multivariable Cox-proportional hazards analyses, each standard deviation (SD) increment in the ratio of ceramides C24:0/C16:0 was associated with a 27% reduction in the risk of dementia (HR 0.73, 95% CI 0.56-0.96) and AD dementia (HR 0.73, 95% CI 0.53-1.00). The ratio of ceramides C22:0/C16:0 was also inversely associated with incident dementia (HR per SD 0.75, 95% CI 0.57-0.98), and approached statistical significance for AD (HR 0.73, 95% CI 0.53-1.01, P = 0.056). Higher ratios of ceramides C24:0/C16:0 and C22:0/C16:0 were also cross-sectionally associated with lower white matter hyperintensity burden on MRI (-0.05 ± 0.02, P = 0.02; -0.06 ± 0.02, P = 0.003; respectively per SD increase), but not with other MRI brain measures. CONCLUSIONS: Higher plasma ratios of very long-chain to long-chain ceramides are associated with a reduced risk of incident dementia and AD dementia in our community-based sample. Circulating ceramide ratios may serve as potential biomarkers for predicting dementia risk in cognitively healthy adults.
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Envejecimiento/sangre , Ceramidas/sangre , Demencia/sangre , Demencia/epidemiología , Leucoaraiosis/sangre , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/epidemiología , Biomarcadores/sangre , Estudios Transversales , Femenino , Humanos , Leucoaraiosis/diagnóstico por imagen , Leucoaraiosis/patología , Estudios Longitudinales , Imagen por Resonancia Magnética , Masculino , Massachusetts/epidemiología , Persona de Mediana Edad , Modelos de Riesgos ProporcionalesRESUMEN
Niemann-Pick disease type C1 (NPC1) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, there is an urgency to improve diagnostics and monitor therapeutic efficacy with biomarkers. In this study, we sought to define the structure of an unknown lipid biomarker for NPC1 with [M + H]+ ion at m/z 509.3351, previously designated as lysoSM-509. The structure of N-palmitoyl-O-phosphocholineserine (PPCS) was proposed for the lipid biomarker based on the results from mass spectrometric analyses and chemical derivatizations. As no commercial standard is available, authentic PPCS was chemically synthesized, and the structure was confirmed by comparison of endogenous and synthetic compounds as well as their derivatives using liquid chromatography-tandem mass spectrometry (LC-MS/MS). PPCS is the most abundant species among N-acyl-O-phosphocholineserines (APCS), a class of lipids that have not been previously detected in biological samples. Further analysis demonstrated that all APCS species with acyl groups ranging from C14 to C24 were elevated in NPC1 plasma. PPCS is also elevated in both central and peripheral tissues of the NPC1 cat model. Identification of APCS structures provide an opportunity for broader exploration of the roles of these novel lipids in NPC1 disease pathology and diagnosis.
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Enfermedad de Niemann-Pick Tipo C/metabolismo , Fosforilcolina/metabolismo , Animales , Biomarcadores/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Enfermedad de Niemann-Pick Tipo C/genéticaRESUMEN
Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPßCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) - comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPßCD/PEG - shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPßCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.
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2-Hidroxipropil-beta-Ciclodextrina/uso terapéutico , Inhibidores de Histona Desacetilasas/uso terapéutico , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Polietilenglicoles/uso terapéutico , Vorinostat/uso terapéutico , Animales , Células Cultivadas , Combinación de Medicamentos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/metabolismoRESUMEN
Cholesterol is an essential structural component of cellular membranes and precursor molecule for oxysterol, bile acid, and hormone synthesis. The study of intracellular cholesterol trafficking pathways has been limited in part due to a lack of suitable cholesterol analogues. Herein, we developed three novel diazirine alkyne cholesterol probes: LKM38, KK174, and KK175. We evaluated these probes as well as a previously described diazirine alkyne cholesterol analogue, trans-sterol, for their fidelity as cholesterol mimics and for study of cholesterol trafficking. LKM38 emerged as a promising cholesterol mimic because it both sustained the growth of cholesterol-auxotrophic cells and appropriately regulated key cholesterol homeostatic pathways. When presented as an ester in lipoprotein particles, LKM38 initially localized to the lysosome and subsequently trafficked to the plasma membrane and endoplasmic reticulum. LKM38 bound to diverse, established cholesterol binding proteins. Through a detailed characterization of the cellular behavior of a panel of diazirine alkyne probes using cell biological, biochemical trafficking assays and immunofluorescence approaches, we conclude that LKM38 can serve as a powerful tool for the study of cholesterol protein interactions and trafficking.
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Alquinos/química , Colesterol/metabolismo , Diazometano/síntesis química , Diazometano/metabolismo , Espacio Intracelular/metabolismo , Sondas Moleculares/síntesis química , Sondas Moleculares/metabolismo , Transporte Biológico , Línea Celular Tumoral , Técnicas de Química Sintética , Diazometano/química , Homeostasis , Humanos , Lipoproteínas/metabolismo , Lisosomas/metabolismo , Sondas Moleculares/químicaRESUMEN
BACKGROUND: Niemann-Pick disease type C1 (NPC1) is a rare, neurodegenerative cholesterol storage disorder. Diagnostic delay of >5â¯years is common due to the rarity of the disease and non-specific early symptoms. To improve diagnosis and facilitate early intervention, we previously developed a newborn screening assay based on newly identified plasma bile acid biomarkers. Because the newborn screen had been validated using dried blood spots (DBS) from already diagnosed NPC1 patients, an unanswered question was whether the screen would be able to detect individuals with NPC1 at birth. METHODS: To address this critical question, we obtained the newborn DBS for already diagnosed NPC1 subjects (nâ¯=â¯15) and carriers (nâ¯=â¯3) residing in California, New York, and Michigan states that archive residual DBS in biorepositories. For each of the DBS, we obtained two neighbor controls - DBS from patients born on the same day and in the same hospital as the NPC1 patients and carriers. 3ß,5α,6ß-trihydroxycholanic acid (bile acid A) and trihydroxycholanic acid glycine conjugate (bile acid B) were measured in the DBS using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. RESULTS: Bile acid B, the more specific biomarker for which the fully validated DBS assay was developed, was detected in 8/15 NPC1 patients, and elevated above the cut-off in 2/15 patients (the two samples with the shortest storage time). Bile acid B was detected in 2/2, 6/10, and 0/7 NPC1 samples that have been stored for <10.5â¯years, 13-20â¯years, andâ¯>â¯20â¯years, respectively, indicating that the glycine conjugate is detectable in DBS but may have reduced long-term stability compared with bile acid A, the precursor trihydroxycholanic acid, which was elevated in 15/15 NPC1 subjects, but not in carriers and controls. CONCLUSIONS: These results demonstrate that newborn screening for NPC1 disease is feasible using bile acid biomarkers.
Asunto(s)
Ácidos y Sales Biliares/análisis , Pruebas con Sangre Seca , Enfermedad de Niemann-Pick Tipo C/sangre , Enfermedad de Niemann-Pick Tipo C/diagnóstico , Bancos de Muestras Biológicas , Biomarcadores/sangre , California , Estudios de Casos y Controles , Cromatografía Liquida , Femenino , Humanos , Recién Nacido , Masculino , Michigan , Tamizaje Neonatal , New York , Estudios Retrospectivos , Espectrometría de Masas en TándemRESUMEN
Small nucleolar RNAs (snoRNAs) are noncoding RNAs that guide chemical modifications of structural RNAs. Whereas snoRNAs primarily localize in the nucleolus, where their canonical function is to target nascent ribosomal RNAs for 2'-O-methylation, recent studies provide evidence that snoRNAs traffic out of the nucleus. Furthermore, RNA-Seq data indicate that extracellular vesicles released from cells contain snoRNAs. However, it is not known whether snoRNA secretion is regulated or whether secreted snoRNAs are functional. Here, we show that inflammation stimulates secretion of Rpl13a snoRNAs U32a (SNORD32a), U33 (SNORD33), U34 (SNORD34), and U35a (SNORD35a) from cultured macrophages, in mice, and in human subjects. Secreted snoRNAs co-fractionate with extracellular vesicles and are taken up by recipient cells. In a murine parabiosis model, we demonstrate that snoRNAs travel through the circulation to function in distant tissues. These findings support a previously unappreciated link between inflammation and snoRNA secretion in mice and humans and uncover a potential role for secreted snoRNAs in cell-cell communication.