RESUMO
The renin-angiotensin-aldosterone system (RAAS) plays a well-characterized role regulating blood pressure in mammals. Pharmacological and genetic manipulation of the RAAS has been shown to extend lifespan in Caenorhabditis elegans, Drosophila and rodents, but its mechanism is not well defined. Here, we investigate the angiotensin-converting enzyme (ACE) inhibitor drug captopril, which extends lifespan in worms and mice. To investigate the mechanism, we performed a forward genetic screen for captopril-hypersensitive mutants. We identified a missense mutation that causes a partial loss of function of the daf-2 receptor tyrosine kinase gene, a powerful regulator of aging. The homologous mutation in the human insulin receptor causes Donohue syndrome, establishing these mutant worms as an invertebrate model of this disease. Captopril functions in C. elegans by inhibiting ACN-1, the worm homolog of ACE. Reducing the activity of acn-1 via captopril or RNA interference promoted dauer larvae formation, suggesting that acn-1 is a daf gene. Captopril-mediated lifespan extension was abrogated by daf-16(lf) and daf-12(lf) mutations. Our results indicate that captopril and acn-1 influence lifespan by modulating dauer formation pathways. We speculate that this represents a conserved mechanism of lifespan control.
Assuntos
Proteínas de Caenorhabditis elegans , Captopril , Animais , Humanos , Camundongos , Captopril/farmacologia , Captopril/metabolismo , Caenorhabditis elegans/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Envelhecimento , Longevidade/fisiologia , Receptor de Insulina/metabolismo , Mutação/genética , Mamíferos/metabolismoRESUMO
De novo lipogenesis is a highly regulated metabolic process, which is known to be activated through transcriptional regulation of lipogenic genes, including fatty acid synthase (FASN). Unexpectedly, we find that the expression of FASN protein remains unchanged during Drosophila larval development from the second to the third instar larval stages (L2 to L3) when lipogenesis is hyperactive. Instead, acetylation of FASN is significantly upregulated in fast-growing larvae. We further show that lysine K813 residue is highly acetylated in developing larvae, and its acetylation is required for elevated FASN activity, body fat accumulation, and normal development. Intriguingly, K813 is autoacetylated by acetyl-CoA (AcCoA) in a dosage-dependent manner independent of acetyltransferases. Mechanistically, the autoacetylation of K813 is mediated by a novel P-loop-like motif (N-xx-G-x-A). Lastly, we find that K813 is deacetylated by Sirt1, which brings FASN activity to baseline level. In summary, this work uncovers a previously unappreciated role of FASN acetylation in developmental lipogenesis and a novel mechanism for protein autoacetylation, through which Drosophila larvae control metabolic homeostasis by linking AcCoA, lysine acetylation, and de novo lipogenesis.
Assuntos
Drosophila , Lipogênese , Animais , Lipogênese/genética , Acetilcoenzima A , Drosophila/genética , Lisina , Ácido Graxo Sintases/genética , Larva/genéticaRESUMO
Parasitic nematodes cause significant morbidity and mortality globally. Excretory/secretory products (ESPs) such as fatty acid- and retinol- binding proteins (FARs) are hypothesized to suppress host immunity during nematode infection, yet little is known about their interactions with host tissues. Leveraging the insect parasitic nematode, Steinernema carpocapsae, we describe here the first in vivo study demonstrating that FARs modulate animal immunity, causing an increase in susceptibility to bacterial co-infection. Moreover, we show that FARs dampen key components of the fly immune response including the phenoloxidase cascade and antimicrobial peptide (AMP) production. Our data also reveal that FARs deplete lipid signaling precursors in vivo as well as bind to these fatty acids in vitro, suggesting that FARs elicit their immunomodulatory effects by altering the availability of lipid signaling molecules necessary for an efficient immune response. Collectively, these data support a complex role for FARs in immunosuppression in animals and provide detailed mechanistic insight into parasitism in phylum Nematoda.
Assuntos
Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Infecções por Nematoides/imunologia , Proteínas de Ligação ao Retinol/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila melanogaster , Nematoides , Infecções por Nematoides/parasitologiaRESUMO
BACKGROUND: Niemann-Pick disease, type C (NPC) is a childhood-onset, lethal, neurodegenerative disorder caused by autosomal recessive mutations in the genes NPC1 or NPC2 and characterized by impaired cholesterol homeostasis, a lipid essential for cellular function. Cellular cholesterol levels are tightly regulated, and mutations in either NPC1 or NPC2 lead to deficient transport and accumulation of unesterified cholesterol in the late endosome/lysosome compartment, and progressive neurodegeneration in affected individuals. Previous cell-based studies to understand the NPC cellular pathophysiology and screen for therapeutic agents have mainly used patient fibroblasts. However, these do not allow modeling the neurodegenerative aspect of NPC disease, highlighting the need for an in vitro system that permits understanding the cellular mechanisms underlying neuronal loss and identifying appropriate therapies. This study reports the development of a novel human iPSC-derived, inducible neuronal model of Niemann-Pick disease, type C1 (NPC1). RESULTS: We generated a null i3Neuron (inducible × integrated × isogenic) (NPC1-/- i3Neuron) iPSC-derived neuron model of NPC1. The NPC1-/- and the corresponding isogenic NPC1+/+ i3Neuron cell lines were used to efficiently generate homogenous, synchronized neurons that can be used in high-throughput screens. NPC1-/- i3Neurons recapitulate cardinal cellular NPC1 pathological features including perinuclear endolysosomal storage of unesterified cholesterol, accumulation of GM2 and GM3 gangliosides, mitochondrial dysfunction, and impaired axonal lysosomal transport. Cholesterol storage, mitochondrial dysfunction, and axonal trafficking defects can be ameliorated by treatment with 2-hydroxypropyl-ß-cyclodextrin, a drug that has shown efficacy in NPC1 preclinical models and in a phase 1/2a trial. CONCLUSION: Our data demonstrate the utility of this new cell line in high-throughput drug/chemical screens to identify potential therapeutic agents. The NPC1-/- i3Neuron line will also be a valuable tool for the NPC1 research community to explore the pathological mechanisms contributing to neuronal degeneration.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Niemann-Pick Tipo C , Colesterol , Humanos , Neurônios , Doença de Niemann-Pick Tipo C/genética , Preparações FarmacêuticasRESUMO
Microdeletions in 19q12q13.12 cause a rare and complex haploinsufficiency syndrome characterized by intellectual deficiency, developmental delays, and neurological movement disorders. Variability in the size and interval of the deletions makes it difficult to attribute the complex clinical phenotype of this syndrome to an underlying gene(s). As an alternate approach, we examined the biochemical and metabolic features of fibroblasts from an affected individual to derive clues as to the molecular basis for the syndrome. Immunofluorescence and electron microscopy of affected fibroblasts revealed an abnormal endo-lysosomal compartment that was characterized by rapid accumulation of lysosomotropic dyes, elevated LAMP1 and LAMP2 expression and vacuoles containing membrane whorls, common features of lysosomal lipid storage disorders. The late endosomes-lysosomes (LE/LY) of affected fibroblasts accumulated low-density lipoprotein cholesterol, and displayed reduced cholesterol esterification and increased de novo cholesterol synthesis, indicative of defective cholesterol transport to the endoplasmic reticulum. Affected fibroblasts also had increased ceramide and sphingolipid mass, altered glycosphingolipid species and accumulation of a fluorescent lactosylceramide probe in LE/LY. Autophagosomes also accumulated in affected fibroblasts because of decreased fusion with autolysosomes, a defect associated with other lysosomal storage diseases. Attempts to correct the cholesterol/sphingolipid storage defect in fibroblasts with cyclodextrin, sphingolipid synthesis inhibitors or by altering ion transport were unsuccessful. Our data show that 19q13.12 deletion fibroblasts have abnormal accumulation of cholesterol and sphingolipids in the endo-lysosomal system that compromises organelle function and could be an underlying cause of the clinical features of the syndrome.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 19/genética , Fibroblastos/patologia , Doenças por Armazenamento dos Lisossomos/patologia , Doenças Raras/patologia , Células Cultivadas , Criança , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Endossomos/patologia , Endossomos/ultraestrutura , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Doenças por Armazenamento dos Lisossomos/diagnóstico , Doenças por Armazenamento dos Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/patologia , Lisossomos/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Doenças Raras/diagnóstico , Doenças Raras/genética , Esfingolipídeos/metabolismo , Síndrome , Sequenciamento Completo do GenomaRESUMO
Diseases of the liver related to metabolic syndrome have emerged as the most common and undertreated hepatic ailments. The cause of nonalcoholic fatty liver disease is the aberrant accumulation of lipid in hepatocytes, though the mechanisms whereby this leads to hepatocyte dysfunction, death, and hepatic fibrosis are still unclear. Insulin-sensitizing thiazolidinediones have shown efficacy in treating nonalcoholic steatohepatitis (NASH), but their widespread use is constrained by dose-limiting side effects thought to be due to activation of the peroxisome proliferator-activated receptor γ. We sought to determine whether a next-generation thiazolidinedione with markedly diminished ability to activate peroxisome proliferator-activated receptor γ (MSDC-0602) would retain its efficacy for treating NASH in a rodent model. We also determined whether some or all of these beneficial effects would be mediated through an inhibitory interaction with the mitochondrial pyruvate carrier 2 (MPC2), which was recently identified as a mitochondrial binding site for thiazolidinediones, including MSDC-0602. We found that MSDC-0602 prevented and reversed liver fibrosis and suppressed expression of markers of stellate cell activation in livers of mice fed a diet rich in trans-fatty acids, fructose, and cholesterol. Moreover, mice with liver-specific deletion of MPC2 were protected from development of NASH on this diet. Finally, MSDC-0602 directly reduced hepatic stellate cell activation in vitro, and MSDC-0602 treatment or hepatocyte MPC2 deletion also limited stellate cell activation indirectly by affecting secretion of exosomes from hepatocytes. CONCLUSION: Collectively, these data demonstrate the effectiveness of MSDC-0602 for attenuating NASH in a rodent model and suggest that targeting hepatic MPC2 may be an effective strategy for pharmacologic development. (Hepatology 2017;65:1543-1556).
Assuntos
Acetofenonas/uso terapêutico , Proteínas de Transporte de Ânions/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Tiazolidinedionas/uso terapêutico , Acetofenonas/farmacologia , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Exossomos/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Distribuição Aleatória , Tiazolidinedionas/farmacologiaRESUMO
Deficiencies of galactosylceramidase and glucocerebrosidase result in the accumulation of galactosylsphingosine (GalSph) and glucosylsphingosine (GluSph) in Krabbe and Gaucher diseases, respectively. GalSph and GluSph are useful biomarkers for both diagnosis and monitoring of treatment effects. We have developed and validated a sensitive, accurate, high-throughput assay for simultaneous determination of the concentration of GalSph and GluSph in mouse serum. GalSph and GluSph and their deuterated internal standards were extracted by protein precipitation in quantitative recoveries, baseline separated by hydrophilic interaction chromatography and detected by positive-ion electrospray mass spectrometry in multiple reaction monitoring mode. Total run time was 7 min. The lower limit of quantification was 0.2 ng/mL for both GalSph and GluSph. Sample stability, assay precision and accuracy, and method robustness were demonstrated. This method has been successfully applied to measurement of these lipid biomarkers in a natural history study in twitcher (Krabbe) mice.
Assuntos
Biomarcadores/sangue , Cromatografia Líquida/métodos , Doença de Gaucher/sangue , Psicosina/análogos & derivados , Psicosina/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Modelos Animais de Doenças , Doença de Gaucher/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Lineares , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
UNLABELLED: Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT: Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Doença de Gaucher/patologia , Glucosilceramidas/antagonistas & inibidores , Glicolipídeos/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Transtornos Parkinsonianos/patologia , alfa-Sinucleína/metabolismo , Acetanilidas/farmacologia , Benzamidas/farmacologia , Catecolaminas/metabolismo , Diferenciação Celular/genética , Neurônios Dopaminérgicos/efeitos dos fármacos , Feminino , Glucosilceramidase , Glucosilceramidas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Mutação/genética , Técnicas de Patch-Clamp , beta-Glucosidase/genéticaRESUMO
Mutations in GBA1 encountered in Gaucher disease are a leading risk factor for Parkinson disease and associated Lewy body disorders. Many GBA1 mutation carriers, especially those with severe or null GBA1 alleles, have earlier and more progressive parkinsonism. To model the effect of partial glucocerebrosidase deficiency on neurological progression in vivo, mice with a human A53T α-synuclein (SNCAA53T) transgene were crossed with heterozygous null gba mice (gba+/-). Survival analysis of 84 mice showed that in gba+/-//SNCAA53T hemizygotes and homozygotes, the symptom onset was significantly earlier than in gba+/+//SNCAA53T mice (p-values 0.023-0.0030), with exacerbated disease progression (p-value <0.0001). Over-expression of SNCAA53T had no effect on glucocerebrosidase levels or activity. Immunoblotting demonstrated that gba haploinsufficiency did not lead to increased levels of either monomeric SNCA or insoluble high molecular weight SNCA in this model. Immunohistochemical analyses demonstrated that the abundance and distribution of SNCA pathology was also unaltered by gba haploinsufficiency. Thus, while the underlying mechanism is not clear, this model shows that gba deficiency impacts the age of onset and disease duration in aged SNCAA53T mice, providing a valuable resource to identify modifiers, pathways and possible moonlighting roles of glucocerebrosidase in Parkinson pathogenesis.
Assuntos
Doença de Gaucher/genética , Glucosilceramidase/genética , Haploinsuficiência , Doença de Parkinson/genética , alfa-Sinucleína/genética , Idade de Início , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Doença de Gaucher/complicações , Glucosilceramidase/deficiência , Glucosilceramidas/análise , Heterozigoto , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Doença de Parkinson/etiologia , Psicosina/análogos & derivados , Psicosina/análise , Transgenes , alfa-Sinucleína/análise , alfa-Sinucleína/deficiência , alfa-Sinucleína/metabolismo , beta-Glucosidase/deficiência , beta-Glucosidase/genéticaRESUMO
Niemann-Pick Type C1 (NPC1) disease is a rare neurovisceral, cholesterol-sphingolipid lysosomal storage disorder characterized by ataxia, motor impairment, progressive intellectual decline, and dementia. The most prevalent mutation, NPC1(I1061T), encodes a misfolded protein with a reduced half-life caused by ER-associated degradation. Therapies directed at stabilization of the mutant NPC1 protein reduce cholesterol storage in fibroblasts but have not been tested in vivo because of lack of a suitable animal model. Whereas the prominent features of human NPC1 disease are replicated in the null Npc1(-/-) mouse, this model is not amenable to examining proteostatic therapies. The objective of the present study was to develop an NPC1 I1061T knock-in mouse in which to test proteostatic therapies. Compared with the Npc1(-/-) mouse, this Npc1(tm(I1061T)Dso) model displays a less severe, delayed form of NPC1 disease with respect to weight loss, decreased motor coordination, Purkinje cell death, lipid storage, and premature death. The murine NPC1(I1061T) protein has a reduced half-life in vivo, consistent with protein misfolding and rapid ER-associated degradation, and can be stabilized by histone deacetylase inhibition. This novel mouse model faithfully recapitulates human NPC1 disease and provides a powerful tool for preclinical evaluation of therapies targeting NPC1 protein variants with compromised stability.
Assuntos
Alelos , Proteínas de Transporte/genética , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Glicoproteínas de Membrana/genética , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Animais , Células Cultivadas , Feminino , Técnicas de Introdução de Genes/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína C1 de Niemann-Pick , PrevalênciaRESUMO
Niemann-Pick C1 (NPC1) disease is a rare, neurodegenerative lysosomal cholesterol storage disorder, typified by progressive cognitive and motor function impairment. Affected individuals usually succumb to the disease in adolescence. 2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) has emerged as a promising intervention that reduces lipid storage and prolongs survival in NPC1 disease animal models. A barrier to the development of HP-ß-CD and other treatments for NPC disease has been the lack of validated biochemical measures to evaluate efficacy. Here we explored whether cholesterol homeostatic responses resulting from HP-ß-CD-mediated redistribution of sequestered lysosomal cholesterol could provide biomarkers to monitor treatment. Upon direct CNS delivery of HP-ß-CD, we found increases in plasma 24(S)-HC in two independent NPC1 disease animal models, findings that were confirmed in human NPC1 subjects receiving HP-ß-CD. Since circulating 24(S)-HC is almost exclusively CNS-derived, the increase in plasma 24(S)-HC provides a peripheral, non-invasive measure of the CNS effect of HP-ß-CD. Our findings suggest that plasma 24(S)-HC, along with the other cholesterol-derived markers examined in this study, can serve as biomarkers that will accelerate development of therapeutics for NPC1 disease.
Assuntos
Colesterol/sangue , Doença de Niemann-Pick Tipo C/tratamento farmacológico , beta-Ciclodextrinas/administração & dosagem , 2-Hidroxipropil-beta-Ciclodextrina , Adolescente , Animais , Biomarcadores/sangue , Criança , Modelos Animais de Doenças , Monitoramento de Medicamentos/métodos , Feminino , Homeostase , Humanos , Masculino , Camundongos Endogâmicos BALB C , Doença de Niemann-Pick Tipo C/sangue , Adulto JovemRESUMO
Niemann-Pick type C (NPC) is a neurodegenerative lysosomal storage disorder caused by defects in the lysosomal proteins NPC1 or NPC2. NPC cells are characterized by reduced lysosomal calcium levels and impaired sphingosine transport from lysosomes. Natural killer (NK) cells kill virally infected/transformed cells via degranulation of lysosome-related organelles. Their trafficking from lymphoid tissues into the circulation is dependent on sphingosine-1-phosphate (S1P) gradients, sensed by S1P receptor 5 (S1P5). We hypothesized that NK-cell function and trafficking could be affected in NPC disease due to the combined effects of the lysosomal calcium defect and sphingosine storage. In an NPC1 mouse model, we found the frequency of NK cells was altered and phenocopied S1P5-deficient mice, consistent with defects in S1P levels. NK cells from NPC1 mice also had a defect in cytotoxicity due to a failure in degranulation of cytotoxic granules, which was associated with reduced lysosomal calcium levels. Affected NPC1 patients and NPC1 heterozygote carriers had reduced NK-cell numbers in their blood and showed similar phenotypic and developmental changes to those observed in the NPC1 mouse. These findings highlight the effects of lysosomal storage on the peripheral immune system.
Assuntos
Células Matadoras Naturais/citologia , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/imunologia , Adolescente , Adulto , Idoso , Animais , Cálcio/metabolismo , Criança , Pré-Escolar , Feminino , Heterozigoto , Humanos , Lactente , Recém-Nascido , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Leucócitos Mononucleares/citologia , Lisofosfolipídeos/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteína C1 de Niemann-Pick , Fenótipo , Proteínas/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Adulto JovemRESUMO
Cholesterol homeostasis is regulated not only by cholesterol, but also by oxygenated cholesterol species, referred to as oxysterols. Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), regulate cholesterol homeostasis through feedback inhibition and feed-forward activation of transcriptional pathways that govern cholesterol synthesis, uptake, and elimination, as well as through direct nongenomic actions that modulate cholesterol accessibility in membranes. Elucidating the cellular distribution of 25-HC is required to understand its biological activity at the molecular level. However, studying oxysterol distribution and behavior within cells has proven difficult due to the lack of fluorescent analogs of 25-HC that retain its chemical and physical properties. To address this, we synthesized a novel intrinsically fluorescent 25-HC mimetic, 25-hydroxycholestatrienol (25-HCTL). We show that 25-HCTL modulates sterol homeostatic responses in a similar manner as 25-HC. 25-HCTL associates with lipoproteins in media and is taken up by cells through LDL-mediated endocytosis. In cultured cells, 25-HCTL redistributes among cellular membranes and, at steady state, has a similar distribution as cholesterol, being enriched in both the endocytic recycling compartment as well as the plasma membrane. Our findings indicate that 25-HCTL is a faithful fluorescent 25-HC mimetic that can be used to investigate the mechanisms through which 25-HC regulates sterol homeostatic pathways.
Assuntos
Corantes Fluorescentes , Hidroxicolesteróis/análise , Animais , Células CHO , Colesterol/análise , Cricetulus , Humanos , Metabolismo dos LipídeosRESUMO
Oxysterols, oxidized metabolites of cholesterol, are endogenous small molecules that regulate lipid metabolism, immune function, and developmental signaling. Although the cell biology of cholesterol has been intensively studied, fundamental questions about oxysterols, such as their subcellular distribution and trafficking pathways, remain unanswered. We have therefore developed a useful method to image intracellular 20(S)-hydroxycholesterol with both high sensitivity and spatial resolution using click chemistry and fluorescence microscopy. The metabolic labeling of cells with an alkynyl derivative of 20(S)-hydroxycholesterol has allowed us to directly visualize this oxysterol by attaching an azide fluorophore through cyclo-addition. Unexpectedly, we found that this oxysterol selectively accumulates in the Golgi membrane using a pathway that is sensitive to ATP levels, temperature, and lysosome function. Although previous models have proposed nonvesicular pathways for the rapid equilibration of oxysterols between membranes, direct imaging of oxysterols suggests that a vesicular pathway is responsible for differential accumulation of oxysterols in organelle membranes. More broadly, clickable alkynyl sterols may represent useful tools for sterol cell biology, both to investigate the functions of these important lipids and to decipher the pathways that determine their cellular itineraries.
Assuntos
Química Click , Corantes Fluorescentes , Complexo de Golgi/metabolismo , Hidroxicolesteróis , Membranas Intracelulares/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Células CHO , Cricetinae , Cricetulus , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Hidroxicolesteróis/síntese química , Hidroxicolesteróis/química , Hidroxicolesteróis/metabolismo , Camundongos , Microscopia de Fluorescência , Células NIH 3T3RESUMO
2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-ß-CD required for clinical trials has been challenging owing to the dispersity of the HP-ß-CD. To support a phase 1 clinical trial for ICV delivery of HP-ß-CD in NPC1 patients, novel methods for quantification of HP-ß-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and "dilute and shoot" procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-ß-CD. The LC-MS/MS methods are â¼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-ß-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-ß-CD in NPC1 patients.
Assuntos
2-Hidroxipropil-beta-Ciclodextrina/sangue , 2-Hidroxipropil-beta-Ciclodextrina/líquido cefalorraquidiano , Espectrometria de Massas/métodos , Cromatografia Líquida/métodos , Feminino , Humanos , MasculinoRESUMO
Macrophages play a key role in host defense and in tissue repair after injury. Emerging evidence suggests that macrophage dysfunction in states of lipid excess can contribute to the development of insulin resistance and may underlie inflammatory complications of diabetes. Ceramides are sphingolipids that modulate a variety of cellular responses including cell death, autophagy, insulin signaling, and inflammation. In this study we investigated the intersection between TLR4-mediated inflammatory signaling and saturated fatty acids with regard to ceramide generation. Primary macrophages treated with lipopolysaccharide (LPS) did not produce C16 ceramide, whereas palmitate exposure led to a modest increase in this sphingolipid. Strikingly, the combination of LPS and palmitate led to a synergistic increase in C16 ceramide. This response occurred via cross-talk at the level of de novo ceramide synthesis in the ER. The synergistic response required TLR4 signaling via MyD88 and TIR-domain-containing adaptor-inducing interferon beta (TRIF), whereas palmitate-induced ceramide production occurred independent of these inflammatory molecules. This ceramide response augmented IL-1ß and TNFα release, a process that may contribute to the enhanced inflammatory response in metabolic diseases characterized by dyslipidemia.
Assuntos
Ceramidas/biossíntese , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Palmitatos/farmacologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Vias Biossintéticas/efeitos dos fármacos , Extratos Celulares , Células Cultivadas , Interleucina-1beta/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Esfingolipídeos/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Niemann-Pick type C (NPC)1 is a rare neurodegenerative disease for which treatment options are limited. A major barrier to development of effective treatments has been the lack of validated biomarkers to monitor disease progression or serve as outcome measures in clinical trials. Using targeted metabolomics to exploit the complex lipid storage phenotype that is the hallmark of NPC1 disease, we broadly surveyed Npc1(-/-) mouse tissues and identified elevated species across multiple sphingolipid classes that increased with disease progression. There was a striking accumulation of sphingoid bases, monohexosylceramides (MCs), and GM2 gangliosides in liver, and sphingoid bases and GM2 and GM3 gangliosides in brain. These lipids were modestly decreased following miglustat treatment, but markedly decreased in response to treatment with 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), two drugs that have shown efficacy in NPC1 animal models. Extending these studies to human subjects led to identification of sphingolipid classes that were significantly altered in the plasma of NPC1 patients. Plasma MCs and ceramides were elevated, whereas sphingoid bases were reduced in NPC1 subjects. Intervention with miglustat in NPC1 patients was accompanied by striking alterations in plasma (reductions in GM1 and GM3 gangliosides) and cerebrospinal fluid (CSF) (increased MCs) sphingolipids. Similar alterations were observed in the CSF from the NPC1 feline model following HP-ß-CD treatment. Our findings suggest that these lipid biomarkers may prove useful as outcome measures for monitoring efficacy of therapy in clinical trials.
Assuntos
Doença de Niemann-Pick Tipo C/sangue , Esfingolipídeos/sangue , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacologia , 1-Desoxinojirimicina/uso terapêutico , 2-Hidroxipropil-beta-Ciclodextrina , Animais , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Gatos , Cromatografia Líquida de Alta Pressão , Avaliação Pré-Clínica de Medicamentos , Feminino , Gangliosídeos/sangue , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Doença de Niemann-Pick Tipo C/diagnóstico , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Especificidade de Órgãos , Esfingolipídeos/líquido cefalorraquidiano , Sulfoglicoesfingolipídeos/sangue , Espectrometria de Massas em Tandem , beta-Ciclodextrinas/farmacologia , beta-Ciclodextrinas/uso terapêuticoRESUMO
SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK1. PF-543 inhibits SphK1 with a K(i) of 3.6 nM, is sphingosine-competitive and is more than 100-fold selective for SphK1 over the SphK2 isoform. In 1483 head and neck carcinoma cells, which are characterized by high levels of SphK1 expression and an unusually high rate of S1P production, PF-543 decreased the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. In contrast with past reports that show that the growth of many cancer cell lines is SphK1-dependent, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 was effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.
Assuntos
Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Pirrolidinas/farmacologia , Esfingosina/análogos & derivados , Sulfonas/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Humanos , Lisofosfolipídeos/sangue , Metanol , Fosforilação , Pirrolidinas/síntese química , Pirrolidinas/metabolismo , Esfingosina/sangue , Esfingosina/metabolismo , Especificidade por Substrato , Sulfonas/síntese química , Sulfonas/metabolismoRESUMO
Brown adipose tissue (BAT) is an important regulator of metabolic homeostasis through its role in adaptive thermogenesis and control of whole-body glucose metabolism. Lipids play multiple roles in BAT functions, including serving as a fuel source for thermogenesis, mediating inter-organelle cross talk, and acting as BAT-derived signaling molecules that influence systemic energy metabolism. Profiling of various lipids in BAT under distinct metabolic states could provide new insights into their roles in the biology of the thermogenic fat. In this chapter, we describe a step-by-step workflow starting from sample preparations to mass spectrometry-based analysis of fatty acids and phospholipids in BAT.
Assuntos
Tecido Adiposo Marrom , Metabolismo Energético , Tecido Adiposo Marrom/metabolismo , Termogênese , Homeostase , Ácidos Graxos/metabolismoRESUMO
The renin-angiotensin-aldosterone system (RAAS) plays a well-characterized role regulating blood pressure in mammals. Pharmacological and genetic manipulation of the RAAS has been shown to extend lifespan in C. elegans , Drosophila , and rodents, but its mechanism is not well defined. Here we investigate the angiotensin-converting enzyme (ACE) inhibitor drug captopril, which extends lifespan in worms and mice. To investigate the mechanism, we performed a forward genetic screen for captopril hypersensitive mutants. We identified a missense mutation that causes a partial loss-of-function of the daf-2 receptor tyrosine kinase gene, a powerful regulator of aging. The homologous mutation in the human insulin receptor causes Donohue syndrome, establishing these mutant worms as an invertebrate model of this disease. Captopril functions in C. elegans by inhibiting ACN-1, the worm homolog of ACE. Reducing the activity of acn-1 via captopril or RNAi promoted dauer larvae formation, suggesting acn-1 is a daf gene. Captopril-mediated lifespan extension xwas abrogated by daf-16(lf) and daf-12(lf) mutations. Our results indicate that captopril and acn-1 control aging by modulating dauer formation pathways. We speculate that this represents a conserved mechanism of lifespan control. Summary Statement: Captopril and acn-1 control aging. By demonstrating they regulate dauer formation and interact with daf genes, including a new DAF-2(A261V) mutant corresponding to a human disease variant, we clarified the mechanism.