RESUMO
Cullin RING E3 ligases (CRL) have emerged as key regulators of disease-modifying pathways and therapeutic targets. Cullin3 (Cul3)-containing CRL (CRL3) has been implicated in regulating hepatic insulin and oxidative stress signaling. However, CRL3 function in liver pathophysiology is poorly defined. Here, we report that hepatocyte Cul3 knockout results in rapid resolution of steatosis in obese mice. However, the remarkable resistance of hepatocyte Cul3 knockout mice to developing steatosis does not lead to overall metabolic improvement but causes systemic metabolic disturbances. Liver transcriptomics analysis identifies that CRL3 inactivation causes persistent activation of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant defense pathway, which also reprograms the lipid transcriptional network to prevent TG storage. Furthermore, global metabolomics reveals that NRF2 activation induces numerous NAD+-consuming aldehyde dehydrogenases to increase the cellular NADH/NAD+ ratio, a redox imbalance termed NADH reductive stress that inhibits the glycolysis-citrate-lipogenesis axis in Cul3 knockout livers. As a result, this NRF2-induced cellular lipid storage defect promotes hepatic ceramide accumulation, elevates circulating fatty acids, and worsens systemic insulin resistance in a vicious cycle. Hepatic lipid accumulation is restored, and liver injury and hyperglycemia are attenuated when NRF2 activation and NADH reductive stress are abolished in hepatocyte Cul3/Nrf2 double-knockout mice. The resistance to hepatic steatosis, hyperglycemia, and NADH reductive stress are observed in hepatocyte Keap1 knockout mice with NRF2 activation. In summary, our study defines a critical role of CRL3 in hepatic metabolic regulation and demonstrates that the CRL3 downstream NRF2 overactivation causes hepatic metabolic maladaptation to obesity and insulin resistance.
Assuntos
Fígado Gorduroso , Hiperglicemia , Resistência à Insulina , Animais , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , NAD/metabolismo , Proteínas Culina/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Camundongos Knockout , LipídeosRESUMO
Therapeutic reduction of hydrophobic bile acids exposure is considered beneficial in cholestasis. The Cyp2c70 KO mice lack hydrophilic muricholic acids and have a human-like hydrophobic bile acid pool resulting in hepatobiliary injury. This study investigates if combining an apical sodium-dependent bile acid transporter inhibitor GSK2330672 (GSK) and fibroblast growth factor-15 (FGF15) overexpression, via simultaneous inhibition of bile acid synthesis and gut bile acid uptake, achieves enhanced therapeutic efficacy in alleviating hepatobiliary injury in Cyp2c70 KO mice. The effects of GSK, adeno-associated virus (AAV)-FGF15, and the combined treatment on bile acid metabolism and cholangiopathy were compared in Cyp2c70 KO mice. In female Cyp2c70 KO mice with more severe cholangiopathy than male Cyp2c70 KO mice, the combined treatment was more effective in reversing portal inflammation, ductular reaction, and fibrosis than AAV-FGF15, while GSK was largely ineffective. The combined treatment reduced bile acid pool by â¼80% compared to â¼50% reduction by GSK or AAV-FGF15, and enriched tauro-conjugated ursodeoxycholic acid in the bile. Interestingly, the male Cyp2c70 KO mice treated with AAV-FGF15 or GSK showed attenuated cholangiopathy and portal fibrosis but the combined treatment was ineffective despite reducing bile acid pool. Both male and female Cyp2c70 KO mice showed impaired gut barrier integrity. AAV-FGF15 and the combined treatment, but not GSK, reduced gut exposure to lithocholic acid and improved gut barrier function. In conclusion, the combined treatment improved therapeutic efficacy against cholangiopathy than either single treatment in the female but not male Cyp2c70 KO mice by reducing bile acid pool size and hydrophobicity.
Assuntos
Colestase , Fígado , Animais , Feminino , Humanos , Camundongos , Ácidos e Sais Biliares/metabolismo , Colestase/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fibrose , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
BACKGROUND: Calcific aortic valve disease (CAVD) is the second leading cause of adult heart diseases. The purpose of this study is to investigate whether miR-101-3p plays a role in the human aortic valve interstitial cells (HAVICs) calcification and the underlying mechanisms. METHODS: Small RNA deep sequencing and qPCR analysis were used to determine changes in microRNA expression in calcified human aortic valves. RESULTS: The data showed that miR-101-3p levels were increased in the calcified human aortic valves. Using cultured primary HAVICs, we demonstrated that the miR-101-3p mimic promoted calcification and upregulated the osteogenesis pathway, while anti-miR-101-3p inhibited osteogenic differentiation and prevented calcification in HAVICs treated with the osteogenic conditioned medium. Mechanistically, miR-101-3p directly targeted cadherin-11 (CDH11) and Sry-related high-mobility-group box 9 (SOX9), key factors in the regulation of chondrogenesis and osteogenesis. Both CDH11 and SOX9 expressions were downregulated in the calcified human HAVICs. Inhibition of miR-101-3p restored expression of CDH11, SOX9 and ASPN and prevented osteogenesis in HAVICs under the calcific condition. CONCLUSION: miR-101-3p plays an important role in HAVIC calcification through regulation of CDH11/SOX9 expression. The finding is important as it reveals that miR-1013p may be a potential therapeutic target for calcific aortic valve disease.
Assuntos
Valvopatia Aórtica , Caderinas , MicroRNAs , Fatores de Transcrição SOX9 , Adulto , Humanos , Valva Aórtica , Valvopatia Aórtica/genética , Células Cultivadas , MicroRNAs/genética , Osteogênese/genética , Transdução de Sinais , Fatores de Transcrição SOX9/genética , Caderinas/genéticaRESUMO
Very low-density lipoprotein receptor (VLDLR) is a multifunctional transmembrane protein. Beyond the function of the full-length VLDLR in lipid transport, the soluble ectodomain of VLDLR (sVLDLR) confers anti-inflammatory and antiangiogenic roles in ocular tissues through inhibition of canonical Wnt signaling. However, it remains unknown how sVLDLR is shed into the extracellular space. In this study, we present the first evidence that a disintegrin and metalloprotease 17 (ADAM17) is responsible for sVLDLR shedding in human retinal pigment epithelium cells using pharmacological and genetic approaches. Among selected proteinase inhibitors, an ADAM17 inhibitor demonstrated the most potent inhibitory effect on sVLDLR shedding. siRNA-mediated knockdown or CRISPR/Cas9-mediated KO of ADAM17 diminished, whereas plasmid-mediated overexpression of ADAM17 promoted sVLDLR shedding. The amount of shed sVLDLR correlated with an inhibitory effect on the Wnt signaling pathway. Consistent with these in vitro findings, intravitreal injection of an ADAM17 inhibitor reduced sVLDLR levels in the extracellular matrix in the mouse retina. In addition, our results demonstrated that ADAM17 cleaved VLDLR only in cells coexpressing these proteins, suggesting that shedding occurs in a cis manner. Moreover, our study demonstrated that aberrant activation of Wnt signaling was associated with decreased sVLDLR levels, along with downregulation of ADAM17 in ocular tissues of an age-related macular degeneration model. Taken together, our observations reveal the mechanism underlying VLDLR cleavage and identify a potential therapeutic target for the treatment of disorders associated with dysregulation of Wnt signaling.
Assuntos
Proteína ADAM17/metabolismo , Degeneração Macular/metabolismo , Receptores de LDL/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Via de Sinalização Wnt , Proteína ADAM17/antagonistas & inibidores , Proteína ADAM17/genética , Animais , Modelos Animais de Doenças , Humanos , Degeneração Macular/genética , Camundongos , Camundongos Knockout , Domínios Proteicos , Receptores de LDL/genéticaRESUMO
Subretinal fibrosis is a key pathological feature in neovascular age-related macular degeneration (nAMD). Previously, we identified soluble very low-density lipoprotein receptor (sVLDLR) as an endogenous Wnt signaling inhibitor. This study investigates whether sVLDLR plays an anti-fibrogenic role in nAMD models, including Vldlr-/- mice and laser-induced choroidal neovascularization (CNV). We found that fibrosis factors including P-Smad2/3, α-SMA, and CTGF were upregulated in the subretinal area of Vldlr-/- mice and the laser-induced CNV model. The antibody blocking Wnt co-receptor LRP6 significantly attenuated the overexpression of fibrotic factors in these two models. Moreover, there was a significant reduction of sVLDLR in the interphotoreceptor matrix (IPM) in the laser-induced CNV model. A transgenic strain (sVLDLR-Tg) with sVLDLR overexpression in the IPM was generated. Overexpression of sVLDLR ameliorated the profibrotic changes in the subretinal area of the laser-induced CNV model. In addition, Wnt and TGF-ß signaling synergistically promoted fibrogenesis in human primary retinal pigment epithelium (RPE) cells. CRISPR/Cas9-mediated LRP6 gene knockout (KO) attenuated this synergistic effect. The disruption of VLDLR expression promoted, while the overexpression of sVLDLR inhibited TGF-ß-induced fibrosis. These findings suggest that overactivated Wnt signaling enhances the TGF-ß pathway in subretinal fibrosis. sVLDLR confers an antifibrotic effect, at least partially, through the inhibition of Wnt signaling and thus, has therapeutic potential for fibrosis.
Assuntos
Neovascularização de Coroide/complicações , Modelos Animais de Doenças , Fibrose/prevenção & controle , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Degeneração Macular/complicações , Receptores de LDL/fisiologia , Epitélio Pigmentado da Retina/patologia , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibrose/etiologia , Fibrose/metabolismo , Fibrose/patologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Epitélio Pigmentado da Retina/metabolismo , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização WntRESUMO
Diabetic retinopathy (DR) is a common complication of diabetes and a leading cause of blindness among the working-age population. Diabetic patients often experience functional deficits in dark adaptation, contrast sensitivity, and color perception before any microvascular pathologies on the fundus become detectable. Previous studies showed that the regeneration of 11-cis-retinal and visual pigment is impaired in a type 1 diabetes animal model, which negatively affects visual function at the early stage of DR. Here, Akita mice, type 1 diabetic model, were treated with the visual pigment chromophore, 9-cis-retinal. This treatment rescued a- and b-wave amplitudes of scotopic electroretinography responses, compared with vehicle-treated Akita mice. In addition, the administration of 9-cis-retinal alleviated oxidative stress significantly as shown by reduced 3-nitrotyrosine levels in the retina of Akita mice. Furthermore, the 9-cis-retinal treatment decreased retinal apoptosis as shown by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and DNA fragment enzyme-linked immunosorbent assay. Overall, these findings showed that 9-cis-retinal administration restored visual pigment formation and decreased oxidative stress and retinal degeneration, which resulted in improved visual function in diabetic mice, suggesting that chromophore deficiency plays a causative role in visual defects in early DR.
Assuntos
Retinopatia Diabética/fisiopatologia , Diterpenos/farmacologia , Retina/efeitos dos fármacos , Retinaldeído/farmacologia , Animais , Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Estresse Oxidativo/efeitos dos fármacos , Retina/fisiopatologiaRESUMO
Endothelial progenitor cells (EPCs) contribute to blood vessel formation. Canonical Wnt signaling plays an important role in physiological and pathological angiogenesis and EPC fate regulation. However, the mechanism for Wnt signaling to regulate EPC fate in neovascularization (NV) has not been clearly defined. Here, we showed that very low-density lipoprotein receptor knockout (Vldlr -/- ) mice, a model of ocular NV induced by Wnt signaling overactivation, have increased EPC numbers in the bone marrow, blood, and retina, as well as an elevated mitochondrial membrane potential indicating higher mitochondrial function of EPCs in the circulation. Isolated EPCs from Vldlr -/- mice showed overactivated Wnt signaling, correlating with increased mitochondrial function, mass, and DNA copy numbers, compared with WT EPCs. Our results also demonstrated that Wnt signaling upregulated mitochondrial biogenesis and function, while inhibiting glycolysis in EPCs, which further decreased EPC stemness and promoted EPCs to a more active state toward differentiation, which may contribute to pathologic vascular formation. Fenofibric acid, an active metabolite of fenofibrate, inhibited Wnt signaling and mitochondrial function in EPCs and decreased EPC numbers in Vldlr -/- mice. It also decreased mitochondrial biogenesis and reactive oxygen species production in Vldlr -/- EPCs, which may be responsible for its therapeutic effect on diabetic retinopathy. These findings demonstrated that Wnt signaling regulates EPC fate through metabolism, suggesting potential application of the EPC metabolic profile as predictor and therapeutic target for neovascular diseases. Stem Cells 2019;37:1331-1343.
Assuntos
Células-Tronco/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Células Progenitoras Endoteliais/metabolismo , Metaboloma , Camundongos , Neovascularização Patológica/metabolismoRESUMO
PURPOSE: Elevated blood levels of C-reactive protein (CRP) are associated with both type 1 and type 2 diabetes and diabetic complications, such as diabetic retinopathy (DR). However, its pathogenic role in DR remains unknown. The present study aims to investigate the potential role of CRP in DR pathogenesis and explore its underlying mechanism. MATERIALS AND METHODS: Human CRP transgenic (hCRP-Tg) rats were employed for streptozotocin (STZ)-induced diabetic and oxygen-induced retinopathy (OIR) models. The retina function was monitored by electroretinography (ERG) and retinal thickness was measured by optical coherence tomography (OCT). TUNEL and cell death ELISA were performed to measure the apoptosis. Oxidative stress was detected by the measurement of reactive oxygen species (ROS) in cells and 3-Nitrotyrosine staining in tissue sections. RESULTS: In non-diabetic condition, hCRP-Tg with elevated hCRP levels in the retinas demonstrated declined ERG responses and decreased retinal thickness. In STZ-induced diabetic condition, overexpression of hCRP deteriorated retinal neurodegeneration as shown by ERG and apoptosis assays. hCRP also exacerbated retinal leukostasis and acellular capillary formation induced by diabetes. In the OIR model, overexpression of hCRP exacerbated retinal neovascularization (NV). In retinal cell lines, hCRP treatment induced cell death and over-production of ROS. Furthermore, hCRP-induced overexpression of pro-inflammatory, pro-oxidative, and pro-angiogenic factors was associated with up-regulation of CD32 and the NF-κB signaling in the retinas. CONCLUSIONS: Elevated hCRP levels play a pathogenic role in DR. Targeting the hCRP-CD32-NF-κB pathway may represent a novel therapeutic strategy for DR.
Assuntos
Proteína C-Reativa/metabolismo , Retinopatia Diabética/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose , Proteína C-Reativa/genética , Retinopatia Diabética/complicações , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Humanos , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Retina/metabolismo , Retina/patologia , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Transdução de SinaisRESUMO
The purpose of this study was to explore the role of microRNA-451a (miR-451a) in diabetic retinopathy through activating transcription factor 2 (ATF2). The epiretinal membrane samples from patients with proliferative diabetic retinopathy (PDR) were immunolabeled with an antibody for Ki-67 to identify the proliferative cells. The expression of miR-451a was measured by qRT-PCR in the retina of Akita mice and in RPE cells under diabetic conditions. The potential downstream targets of miR-451a were predicted by bioinformatics and confirmed by dual luciferase assay, qRT-PCR, and Western blotting. Mitochondrial function, cell proliferation, and migration assays were used to detect the functional change after transfection of miR-451a mimic and inhibitor. Proliferative RPE cells were identified in the epiretinal membrane from PDR patients. The expression of miR-451a was downregulated both in the retina of Akita mice and 4-hydroxynonenal (4-HNE)-treated RPE cells. Bioinformatic analysis and luciferase assay identified ATF2 as a potential target of miR-451a. miR-451a inhibited proliferation and migration of RPE cells. The mitochondrial function was enhanced by miR-451a mimic, but suppressed by miR-451a inhibitor. In diabetic conditions, miR-451a showed a protective effect on mitochondrial function. The results of qRT-PCR and Western blotting revealed that overexpression of miR-451a downregulated the expression of ATF2 and its downstream target genes CyclinA1, CyclinD1, and MMP2. In conclusion, miR-451a/ATF2 plays a vital role in the regulation of proliferation and migration in RPE cells through regulation of mitochondrial function, which may provide new perspectives for developing effective therapies for PDR.
Assuntos
Fator 2 Ativador da Transcrição/genética , Retinopatia Diabética/genética , MicroRNAs/genética , Mitocôndrias/metabolismo , Fator 2 Ativador da Transcrição/metabolismo , Adulto , Idoso , Animais , Movimento Celular , Proliferação de Células , Ciclina A1/genética , Ciclina A1/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Retinopatia Diabética/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Epitélio Pigmentado da RetinaRESUMO
Human apolipoprotein E (apoE) is one of the major determinants in lipid transport, playing a critical role in atherosclerosis and other diseases. Binding to lipid and heparan sulfate proteoglycans (HSPG) induces apoE to adopt active conformations for binding to low-density lipoprotein receptor (LDLR) family. ApoE also interacts with beta amyloid peptide, manifests critical isoform-specific effects on Alzheimer's disease. Despite the importance of apoE in these major human diseases, the fundamental questions of how apoE adjusts its structure upon binding to regulate its diverse functions remain unsolved. We report the NMR structure of apoE3, displaying a unique topology of three structural domains. The C-terminal domain presents a large exposed hydrophobic surface that likely initiates interactions with lipids, HSPG, and beta amyloid peptides. The unique topology precisely regulates apoE tertiary structure to permit only one possible conformational adaptation upon binding and provides a double security in preventing lipid-free and partially-lipidated apoE from premature binding to apoE receptors during receptor biogenesis. This topology further ensures the optimal receptor-binding activity by the fully lipidated apoE during lipoprotein transport in circulation and in the brain. These findings provide a structural framework for understanding the structural basis of the diverse functions of this important protein in human diseases.
Assuntos
Apolipoproteína E3/química , Amiloide/química , Amiloide/metabolismo , Apolipoproteína E3/metabolismo , Transporte Biológico/fisiologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metabolismo dos Lipídeos/fisiologia , Lipídeos/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Liver is a major organ that metabolizes sulfur amino acids cysteine, which is the substrate for the synthesis of many essential cellular molecules including GSH, taurine, and coenzyme A. Bile acid-activated farnesoid x receptor (FXR) inhibits cysteine dioxygenase type 1 (CDO1), which mediates hepatic cysteine catabolism and taurine synthesis. To define the impact of bile acid inhibition of CDO1 on hepatic sulfur amino acid metabolism and antioxidant capacity, we developed hepatocyte-specific CDO1 knockout mice (Hep-CDO1 KO) and hepatocyte specific CDO1 transgenic mice (Hep-CDO1 Tg). Liver metabolomics revealed that genetic deletion of hepatic CDO1 reduced de novo taurine synthesis but had no impact on hepatic taurine abundance or bile acid conjugation. Consistent with reduced cysteine catabolism, Hep-CDO1 KO mice showed increased hepatic cysteine abundance but unaltered methionine cycle intermediates and coenzyme A synthesis. Upon acetaminophen overdose, Hep-CDO1 KO mice showed increased GSH synthesis capacity and alleviated liver injury. In contrast, hepatic CDO1 overexpression in Hep-CDO1 Tg mice stimulated hepatic cysteine to taurine conversion, resulting in reduced hepatic cysteine abundance. However, Hep-CDO1 Tg mice and WT showed similar susceptibility to acetaminophen-induced liver injury. Hep-CDO1 Tg mice showed similar hepatic taurine and coenzyme A compared to WT mice. In summary, these findings suggest that bile acid and FXR signaling inhibition of CDO1-mediated hepatic cysteine catabolism preferentially modulates hepatic GSH synthesis capacity and antioxidant defense, but has minimal effect on hepatic taurine and coenzyme A abundance. Repression of hepatic CDO1 may contribute to the hepatoprotective effects of FXR activation under certain pathologic conditions.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Cisteína Dioxigenase , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Glutationa , Animais , Camundongos , Acetaminofen/metabolismo , Acetaminofen/toxicidade , Antioxidantes/farmacologia , Ácidos e Sais Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Coenzima A/metabolismo , Cisteína/metabolismo , Cisteína Dioxigenase/genética , Cisteína Dioxigenase/metabolismo , Glutationa/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Taurina/farmacologia , Taurina/metabolismoRESUMO
Cyp2c70 knockout mice lack the enzyme that produces muricholic acids and show a "human-like" hydrophobic bile acid pool-induced hepatobiliary injury. In this study, we investigated the potential anti-cholestasis effect of glycine-conjugated ß muricholic acid (G-ß-MCA) in male Cyp2c70 KO mice based on its hydrophilic physiochemical property and signaling property as an farnesoid X receptor (FXR) antagonist. Our results showed that G-ß-MCA treatment for 5 weeks alleviated ductular reaction and liver fibrosis and improved gut barrier function. Analysis of bile acid metabolism suggested that exogenously administered G-ß-MCA was poorly absorbed in the small intestine and mostly deconjugated in the large intestine and converted to taurine-conjugated MCA (T-MCA) in the liver, leading to T-MCA enrichment in the bile and small intestine. These changes decreased the biliary and intestine bile acid hydrophobicity index. Furthermore, G-ß-MCA treatment decreased intestine bile acid absorption via unknown mechanisms, resulting in increased fecal bile acid excretion and a reduction in total bile acid pool size. In conclusion, G-ß-MCA treatment reduces the bile acid pool size and hydrophobicity and improves liver fibrosis and gut barrier function in Cyp2c70 KO mice.
Assuntos
Ácidos e Sais Biliares , Glicina , Camundongos , Masculino , Humanos , Animais , Camundongos Knockout , Glicina/farmacologia , Cirrose Hepática/tratamento farmacológicoRESUMO
Herein, a soft-template strategy involving the cationic surfactants has been successfully applied to size-controlled synthesis of hierarchical porous Fe-N/C for the first time. Specifically, a small amount of Fe and cationic surfactants can be uniformly doped into the zinc-based zeolite imidazole framework (ZIF-8) crystal particles and the cationic surfactants play a critical role in the formation of hierarchically porous Fe-ZIF-8@surfactant precursors. When the Fe-ZIF-8@surfactant is subsequently pyrolyzed, atomically dispersed Fe-Nx coordination structures can be in-situ converted to Fe-N/C, while the cationic surfactants decompose to form a carbon matrix to encapsulate the active sites, thereby preventing the aggregation of nanoparticles to a certain extent. As a result, the combined Fe nanocrystals and atomically dispersed Fe-Nx in the graphitic carbon matrix generate a synergistic effect to boost the electrocatalytic behaviors with a more positive half-wave potential (0.92 V) for oxygen reduction reaction (ORR) and a lower overpotential (420 mV at 10 mA cm-2) for oxygen evolution reaction (OER). As a proof of concept, the Fe-N/C@TTAB based zinc-air batteries (ZABs) present an outstanding peak power density (107.9 mW cm-2) and a superior specific capacity (706.3 mAh g-1) with robust cycling stability over 900 cycles for 150 h, which are better than the commercial Pt/C + IrO2 based ZABs.
RESUMO
Fatty liver is a highly heterogenous condition driven by various pathogenic factors in addition to the severity of steatosis. Protein insufficiency has been causally linked to fatty liver with incompletely defined mechanisms. Here we report that fatty liver is a sulfur amino acid insufficient state that promotes metabolic inflexibility via limiting coenzyme A availability. We demonstrate that the nutrient-sensing transcriptional factor EB synergistically stimulates lysosome proteolysis and methionine adenosyltransferase to increase cysteine pool that drives the production of coenzyme A and glutathione, which support metabolic adaptation and antioxidant defense during increased lipid influx. Intriguingly, mice consuming an isocaloric protein-deficient Western diet exhibit selective hepatic cysteine, coenzyme A and glutathione deficiency and acylcarnitine accumulation, which are reversed by cystine supplementation without normalizing dietary protein intake. These findings support a pathogenic link of dysregulated sulfur amino acid metabolism to metabolic inflexibility that underlies both overnutrition and protein malnutrition-associated fatty liver development.
Assuntos
Aminoácidos Sulfúricos , Fígado Gorduroso , Aminoácidos Sulfúricos/metabolismo , Animais , Antioxidantes/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Coenzima A/metabolismo , Cisteína/metabolismo , Cistina/metabolismo , Proteínas Alimentares/metabolismo , Fígado Gorduroso/metabolismo , Glutationa/metabolismo , Homeostase , Lipídeos , Fígado/metabolismo , Metionina/metabolismo , Metionina Adenosiltransferase/metabolismo , Camundongos , OxirreduçãoRESUMO
NMR structural determination of large multi-domain proteins is a challenging task due to significant spectral overlap with a particular difficulty in unambiguous identification of domain-domain interactions. Segmental labeling is a NMR strategy that allows for isotopically labeling one domain and leaves the other domain unlabeled. This significantly simplifies spectral overlaps and allows for quick identification of domain-domain interaction. Here, a novel segmental labeling strategy is presented for detection of inter-domain NOEs. To identify domain-domain interactions in human apolipoprotein E (apoE), a multi-domain, 299-residues α-helical protein, on-column expressed protein ligation was utilized to generate a segmental-labeled apoE samples in which the N-terminal (NT-) domain was (2)H(99%)/(15)N-labeled whereas the C-terminal (CT-) domain was either (15)N- or (15)N/(13)C-labeled. 3-D (15)N-edited NOESY spectra of these segmental-labeled apoE samples allow for direct observation of the inter-domain NOEs between the backbone amide protons of the NT-domain and the aliphatic protons of the CT-domain. This straightforward approach permits unambiguous identification of 78 inter-domain NOEs, enabling accurate definition of the relative positions of both the NT- and the CT-domains and determination of the NMR structure of apoE.
Assuntos
Apolipoproteínas E/química , Marcação por Isótopo/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Domínios e Motivos de Interação entre Proteínas , Apolipoproteínas E/metabolismo , Sítios de Ligação , Humanos , Modelos Moleculares , Isótopos de NitrogênioRESUMO
Patients with diabetes often experience visual defects before any retinal pathologies are detected. The molecular mechanism for the visual defects in early diabetes has not been elucidated. Our previous study reported that in early diabetic retinopathy (DR), rhodopsin levels were reduced due to impaired 11-cis-retinal regeneration. Interphotoreceptor retinol-binding protein (IRBP) is a visual cycle protein and important for 11-cis-retinal generation. IRBP levels are decreased in the vitreous and retina of DR patients and animal models. To determine the role of IRBP downregulation in the visual defects in early DR, we induced diabetes in transgenic mice overexpressing IRBP in the retina. IRBP overexpression prevented diabetes-induced decline of retinal function. Furthermore, IRBP overexpression also prevented decreases of rhodopsin levels and 11-cis-retinal generation in diabetic mice. Diabetic IRBP transgenic mice also showed ameliorated retinal oxidative stress, inflammation, apoptosis, and retinal degeneration compared with diabetic wild-type mice. These findings suggest that diabetes-induced IRBP downregulation impairs the regeneration of 11-cis-retinal and rhodopsin, leading to retinal dysfunction in early DR. Furthermore, increased 11-cis-retinal-free opsin constitutively activates the phototransduction pathway, leading to increased oxidative stress and retinal neurodegeneration. Therefore, restored IRBP expression in the diabetic retina may confer a protective effect against retinal degeneration in DR.
Assuntos
Retinopatia Diabética/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Rodopsina/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Retinopatia Diabética/genética , Proteínas do Olho/genética , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Proteínas de Ligação ao Retinol/genética , Rodopsina/genética , Tomografia de Coerência ÓpticaRESUMO
Purpose: The retina is a commonly used model for angiogenesis research due to its special characteristics. Oxygen-induced retinopathy (OIR) provides a useful model to study ischemia-induced neovascularization (NV) and to develop anti-angiogenic therapeutics. The purpose of this study was to develop a simple, accurate, and less-subjective quantification method for retinal NV in the OIR model. Methods: To address this challenge, we combined the conventional vascular staining and BrdU labeling of newly formed vascular cells to detect and analyze retinal NV. With daily injections of BrdU, which was incorporated into the DNA of newly formed retinal vessels under the OIR condition, ischemia-induced retinal neovasculature with BrdU labeling was distinguished from pre-existing vasculature and accurately quantified using the ImageJ program. Results: Compared with conventional quantification methods using isolectin B4 staining of the entire vascular network, BrdU labeling allowed us to distinguish newly formed vessels from the pre-existing vessels and to objectively quantify the newly formed vessels, which was verified in OIR mice with intravitreal injections of an antibody-neutralizing vascular endothelial growth factor. Conclusions: BrdU labeling provides a useful and sensitive method for studying retinal NV and evaluating the therapeutic effects of medical interventions against pathological angiogenesis. Translational Relevance: Quantitative, straightforward, and objective observation and evaluation of pathologic neovasculature are important to study the pathogenesis of NV and therapeutic effects using animal models.
Assuntos
Neovascularização Retiniana , Animais , Bromodesoxiuridina , Modelos Animais de Doenças , Camundongos , Vasos Retinianos , Fator A de Crescimento do Endotélio VascularRESUMO
Deficiency of endothelial progenitor cells, including endothelial colony-forming cells (ECFCs) and circulating angiogenic cells (CACs), plays an important role in retinal vascular degeneration in diabetic retinopathy (DR). Fenofibrate, an agonist of peroxisome proliferator-activated receptor α (PPARα), has shown therapeutic effects on DR in both patients and diabetic animal models. However, the function of PPARα in ECFC/CACs has not been defined. In this study, we determined the regulation of ECFC/CAC by PPARα. As shown by flow cytometry and Seahorse analysis, ECFC/CAC numbers and mitochondrial function were decreased in the bone marrow, circulation, and retina of db/db mice, correlating with PPARα downregulation. Activation of PPARα by fenofibrate normalized ECFC/CAC numbers and mitochondrial function in diabetes. In contrast, PPARα knockout exacerbated ECFC/CAC number decreases and mitochondrial dysfunction in diabetic mice. Primary ECFCs from PPARα -/- mice displayed impaired proliferation, migration, and tube formation. Furthermore, PPARα -/- ECFCs showed reduced mitochondrial oxidation and glycolysis compared with wild type, correlating with decreases of Akt phosphorylation and expression of its downstream genes regulating ECFC fate and metabolism. These findings suggest that PPARα is an endogenous regulator of ECFC/CAC metabolism and cell fate. Diabetes-induced downregulation of PPARα contributes to ECFC/CAC deficiency and retinal vascular degeneration in DR.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/metabolismo , Células Progenitoras Endoteliais/metabolismo , PPAR alfa/metabolismo , Retina/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células Progenitoras Endoteliais/efeitos dos fármacos , Fenofibrato/farmacologia , Hipolipemiantes/farmacologia , Masculino , Camundongos , Camundongos Knockout , PPAR alfa/genética , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Retina/efeitos dos fármacosRESUMO
Senescence-accelerated mice P1 (SAMP1) is an aging model characterized by shortened lifespan and early signs of senescence. Klotho is an aging-suppressor gene. The purpose of this study is to investigate whether in vivo expression of secreted klotho (Skl) gene attenuates aortic valve fibrosis in SAMP1 mice. SAMP1 mice and age-matched (AKR/J) control mice were used. SAMP1 mice developed obvious fibrosis in aortic valves, namely fibrotic aortic valve disease. Serum level of Skl was decreased drastically in SAMP1 mice. Expression of MCP-1 (monocyte chemoattractant protein 1), ICAM-1 (intercellular adhesion molecule 1), F4/80, and CD68 was increased in aortic valves of SAMP1 mice, indicating inflammation. An increase in expression of α-smooth muscle actin (myofibroblast marker), transforming growth factorß-1, and scleraxis (a transcription factor of collagen synthesis) was also found in aortic valves of SAMP1 mice, suggesting that accelerated aging is associated with myofibroblast transition and collagen gene activation. We constructed adeno-associated virus 2 carrying mouse Skl cDNA for in vivo expression of Skl. Skl gene delivery effectively increased serum Skl of SAMP1 mice to the control level. Skl gene delivery inhibited inflammation and myofibroblastic transition in aortic valves and attenuated fibrotic aortic valve disease in SAMP1 mice. It is concluded that senescence-related fibrotic aortic valve disease in SAMP1 mice is associated with a decrease in serum klotho leading to inflammation, including macrophage infiltration and transforming growth factorß-1/scleraxis-driven myofibroblast differentiation in aortic valves. Restoration of serum Skl levels by adeno-associated virus 2 carrying mouse Skl cDNA effectively suppresses inflammation and myofibroblastic transition and attenuates aortic valve fibrosis. Skl may be a potential therapeutic target for fibrotic aortic valve disease.
Assuntos
Envelhecimento/genética , Estenose da Valva Aórtica/genética , Regulação da Expressão Gênica , Cardiopatias Congênitas/genética , Doenças das Valvas Cardíacas/genética , Proteínas de Membrana/genética , Análise de Variância , Animais , Valva Aórtica/patologia , Estenose da Valva Aórtica/patologia , Doença da Válvula Aórtica Bicúspide , Biópsia por Agulha , Modelos Animais de Doenças , Regulação para Baixo/genética , Fibrose/genética , Fibrose/patologia , Cardiopatias Congênitas/patologia , Doenças das Valvas Cardíacas/patologia , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/genética , Proteínas Klotho , Longevidade/genética , Camundongos , Camundongos Endogâmicos , Distribuição Aleatória , Sensibilidade e Especificidade , Rigidez Vascular/genéticaRESUMO
Klotho was originally discovered as an aging-suppressor gene. The objective of this study is to investigate whether klotho gene deficiency affects high-fat diet (HFD)-induced arterial stiffening. Heterozygous Klotho-deficient (KL(+/-)) mice and WT littermates were fed on HFD or normal diet. HFD increased pulse wave velocity within 5 weeks in KL(+/-) mice but not in wild-type mice, indicating that klotho deficiency accelerates and exacerbates HFD-induced arterial stiffening. A greater increase in blood pressure was found in KL(+/-) mice fed on HFD. Protein expressions of phosphorylated AMP-activated protein kinase-α (AMPKα), phosphorylated endothelial nitric oxide synthase (eNOS), and manganese-dependent superoxide dismutase (Mn-SOD) were decreased, whereas protein expressions of collagen I, transforming growth factor-ß1, and Runx2 were increased in aortas of KL(+/-) mice fed on HFD. Interestingly, daily injections of an AMPKα activator, 5-aminoimidazole-4-carboxamide-3-ribonucleoside, abolished the increases in pulse wave velocity, blood pressure, and blood glucose in KL(+/-) mice fed on HFD. Treatment with 5-aminoimidazole-4-carboxamide-3-ribonucleoside for 2 weeks not only abolished the downregulation of phosphorylated AMPKα, phosphorylated eNOS, and Mn-SOD levels but also attenuated the increased levels of collagen I, transforming growth factor-ß1, Runx2, superoxide, elastic lamellae breaks, and calcification in aortas of KL(+/-) mice fed on HFD. In cultured mouse aortic smooth muscle cells, cholesterol plus KL-deficient serum decreased phosphorylation levels of AMPKα and LKB1 (an important upstream regulator of AMPKα activity) but increased collagen I synthesis, which can be eliminated by activation of AMPKα by 5-aminoimidazole-4-carboxamide-3-ribonucleoside. In conclusions, Klotho deficiency promoted HFD-induced arterial stiffening and hypertension via downregulation of AMPKα activity.