RESUMO
Intestinal lipid absorption as well as secretion of absorbed lipids as chylomicrons by the enterocytes is a direct measure of the availability of dietary lipids. Measurement of this parameter is central to the understanding of the influence of diet on plasma lipids, specifically when modulation of intestinal lipid absorption by targeted interventions is being examined. In the post-prandial state, very low-density lipoprotein (VLDL) secreted from the liver represent the major source of plasma lipids and rate of VLDL secretion reports on hepatic lipid homeostasis. Here, we describe the methods to specifically measure secretion of chylomicron and VLDL in vivo. Tight regulation of dietary lipid absorption (chylomicron secretion) and hepatic secretion of VLDL underlies the development of dyslipidemia preceding hepatic lipid accumulation seen in non-alcoholic fatty liver disease (NAFLD) and subsequent progression to non-alcoholic steatohepatitis (NASH) underscoring the importance of measurement of lipoprotein secretion in vivo.
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Quilomícrons , Lipoproteínas VLDL , Gorduras na Dieta , Lipoproteínas , Fígado , TriglicerídeosRESUMO
Neutrophil extracellular traps increase cystic fibrosis (CF) airway inflammation. We hypothesized that macrophage exposure to neutrophil elastase (NE) would trigger the release of macrophage extracellular traps (METs), a novel mechanism to augment NE-induced airway inflammation in CF. Experiments were performed using human blood monocyte derived macrophages (hBMDM) from patients with and without CF to test specific mechanisms associated with MET release, and MET release by NE was confirmed in alveolar macrophages from Cftr-null and wild-type littermate mice exposed to intratracheal NE in vivo. Human BMDM were exposed to FITC-NE, and intracellular FITC-NE was localized to cytoplasmic and nuclear domains. Intracellular NE was proteolytically active as indicated by DQ-Elastin substrate cleavage. NE (100 to 500 nM) significantly increased extracellular PicoGreen fluorescence consistent with DNA release/ MET release from hBMDM in the absence of cell death. MET release was further confirmed by confocal microscopy in hBMDM treated with NE, and in alveolar macrophages from Cftr-null and wild-type littermate mice that had been exposed to intratracheal NE. NE-triggered MET release was associated with H3 citrullination detected by immunofluorescence assays and with partial cleavage of histone H3 but not H4. Exposure to NE caused release of METs from both CF and non-CF hBMDM in vitro and murine alveolar macrophages in vivo. MET release was associated with NE-activated H3 clipping, a mechanism associated with chromatin decondensation, a prerequisite for METs.
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Fibrose Cística/metabolismo , Armadilhas Extracelulares/metabolismo , Elastase de Leucócito/metabolismo , Macrófagos/metabolismo , Adulto , Animais , Lavagem Broncoalveolar , Citrulinação , Fibrose Cística/patologia , DNA/metabolismo , Feminino , Histonas/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteólise , Adulto JovemRESUMO
Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE), a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte-derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of Escherichia coli bio-particles. We performed liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and downregulated 211 proteins. NE released not only cell surface proteins into the media but also cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors [including lipoprotein receptor-related protein 1 (LRP1)], 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/secretome included proteins from other macrophage cellular domains, suggesting that NE may globally regulate macrophage structure and function.
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Elastase de Leucócito/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Fagocitose , Fagossomos/metabolismo , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Humanos , Elastase de Leucócito/genética , Lisossomos/genética , Lisossomos/patologia , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Mutantes , Fagossomos/genética , Fagossomos/patologia , Células RAW 264.7RESUMO
[Figure: see text].
Assuntos
Fosfatase Alcalina/sangue , Aterosclerose/metabolismo , Absorção Intestinal , Lipopolissacarídeos/sangue , Animais , Aterosclerose/etiologia , Aterosclerose/patologia , Antígenos CD36/metabolismo , Proteínas de Transporte/metabolismo , Colo/anatomia & histologia , Colo/metabolismo , Dieta Aterogênica/efeitos adversos , Proteínas de Transporte de Ácido Graxo/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas Ligadas por GPI/sangue , Humanos , Mucosa Intestinal , Metabolismo dos Lipídeos , Lipídeos/análise , Lipídeos/sangue , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Redução de PesoAssuntos
Doenças Cardiovasculares , Proteína HMGB1 , Humanos , Inflamação , Estudos Longitudinais , Obesidade , Fatores de Risco , Adulto JovemRESUMO
Reduction of lipoprotein uptake by macrophages and stimulation of cholesterol efflux are two essential steps required for atherosclerotic plaque regression. We used the optimized mannose-functionalized dendrimeric nanoparticle (mDNP)-based platform for macrophage-specific delivery of therapeutics to simultaneously deliver SR-A siRNA (to reduce LDL uptake) and LXR ligand (LXR-L, to stimulate cholesterol efflux) - a novel "Two-pronged" approach to facilitate plaque regression. mDNP-mediated delivery of SR-A siRNA led to a significant reduction in SR-A expression with a corresponding decrease in uptake of oxLDL. Delivery of LXR-L increased expression of ABCA1/G1 and cholesterol efflux. Combined delivery of siRNA and LXR-L led to a significantly greater decrease in macrophage cholesterol content compared to either treatment alone. Administration of this in vitro optimized formulation of mDNP complexed with SR-A-siRNA and LXR-L (Two-pronged complex) to atherosclerotic LDLR-/- mice fed western diet (TD88137) led to significant regression of atherosclerotic plaques with a corresponding decrease in aortic cholesterol content.
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Aterosclerose , Nanopartículas , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Aterosclerose/tratamento farmacológico , Colesterol , Receptores X do Fígado , Camundongos , Camundongos KnockoutRESUMO
INTRODUCTION: Although chronic kidney disease (CKD) is associated with increased risk for coronary artery disease (CAD), the underlying mechanisms are not completely defined. In the present study, we tested the hypothesis that flux of cholesterol from macrophage foam cells to liver is impaired in subjects with CKD. METHODS: Consecutive healthy patients, patients with at least 1 CAD risk factor, patients with established CAD, and patients with CKD stages G3 to G5 (n ≥ 15/group) were recruited prospectively. The ability of total patient serum without any modifications to (i) facilitate efflux of cholesterol from human THP1-macrophage foam cells under physiological conditions (cholesterol efflux capacity [CEC]) and (ii) to deliver this effluxed cholesterol to primary hepatocytes with physiological expression of high-density lipoprotein (HDL) receptor SR-BI (capacity to deliver cholesterol to hepatocytes [CDCH]) was evaluated. RESULTS: Although healthy patients, patients with at least 1 CAD risk factor, and patients with established CAD all showed similar CEC, patients with CKD showed significantly higher CEC. CDCH was significantly lower in all groups compared with the healthy patients; however, when corrected for higher CEC, CDCH in patients with CKD was significantly lower than in patients with CAD. CDCH correlated with age, body mass index, metabolic parameters, inflammatory markers, and kidney function markers (estimated glomerular filtration rate [eGFR], serum creatinine, and serum cystatin C). CONCLUSIONS: These results suggest that aberrations in delivery of cholesterol effluxed from macrophage foam cells to liver for final elimination or the last step of reverse cholesterol transport, may underlie the increased risk of CAD in patients with CKD.
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The intestinal barrier is complex and consists of multiple layers, and it provides a physical and functional barrier to the transport of luminal contents to systemic circulation. While the epithelial cell layer and the outer/inner mucin layer constitute the physical barrier and are often referred to as the intestinal barrier, intestinal alkaline phosphatase (IAP) produced by epithelial cells and antibacterial proteins secreted by Panneth cells represent the functional barrier. While antibacterial proteins play an important role in the host defense against gut microbes, IAP detoxifies bacterial endotoxin lipopolysaccharide (LPS) by catalyzing the dephosphorylation of the active/toxic Lipid A moiety, preventing local inflammation as well as the translocation of active LPS into systemic circulation. The causal relationship between circulating LPS levels and the development of multiple diseases underscores the importance of detailed examination of changes in the "layers" of the intestinal barrier associated with disease development and how this dysfunction can be attenuated by targeted interventions. To develop targeted therapies for improving intestinal barrier function, it is imperative to have a deeper understanding of the intestinal barrier itself, the mechanisms underlying the development of diseases due to barrier dysfunction (eg, high circulating LPS levels), the assessment of intestinal barrier function under diseased conditions, and of how individual layers of the intestinal barrier can be beneficially modulated to potentially attenuate the development of associated diseases. This review summarizes the current knowledge of the composition of the intestinal barrier and its assessment and modulation for the development of potential therapies for barrier dysfunction-associated diseases.
RESUMO
BACKGROUND: A Western-type diet (WD), rich in fat and cholesterol but deficient in fiber, induces development of diabetes and atherosclerosis. Colonic bacteria use the gut's mucous lining as an alternate energy source during periods of fiber deficiency, resulting in intestinal barrier erosion. OBJECTIVE: We hypothesized that supplementing a WD with galactooligosaccharide (GOS) fiber would attenuate WD-induced mucin layer disruption and attenuate development of metabolic diseases. METHODS: C57BL/6 mice (both sexes, 8-10 wk of age) were fed a standard rodent diet (TD7012, reference) or a high-fat, high-cholesterol-containing WD (TD88137, 21% fat, 0.15% cholesterol, 19.5% caesin) or a WD supplemented with 5% GOS fiber (TD170432, WD + GOS) for 16 wk. WD-fed mice that were gavaged daily with curcumin (100 mg/kg) served as positive controls. Glucose tolerance, colonic mucin layer, gene expression, and circulating macrophage/neutrophil levels were determined. Hyperlipidemic Ldlr-/- mice (both sexes, 8-10 wk of age) fed a WD with or without GOS supplementation (for 16 wk) were used to assess plasma LPS and atherosclerosis. Effects of dietary supplementation on different parameters were compared for each genotype. RESULTS: Compared with a WD, glucose tolerance was significantly improved in male C57BL/6 mice fed a WD + GOS (mean ± SEM: AUC = 53.6 ± 43.9 compared with 45.4 ± 33.3 g â min/dL; P = 0.015). Continuity of colonic mucin layer (MUC-2 expression) was improved in mice receiving GOS supplementation, indicating improved intestinal barrier. GOS supplementation also reduced circulating macrophages (30% decrease) and neutrophils (60% decrease), suggesting diminished systemic inflammation. In Ldlr-/- mice, GOS supplementation significantly reduced plasma LPS concentrations (mean ± SEM: 0.81 ± 0.43 EU/mL compared with 0.32 ± 0.26 EU/mL, P < 0.0001, in females and 0.56 ± 0.24 EU/mL compared with 0.34 ± 0.12 EU/mL, P = 0.036, in males), improved glucose tolerance in male mice, and attenuated atherosclerotic lesion area (mean ± SEM: 54.2% ± 6.19% compared with 43.0% ± 35.12%, P = 0.0006, in females and 54.6% ± 3.99% compared with 43.1% ± 8.11%, P = 0.003, in males). CONCLUSIONS: GOS fiber supplementation improves intestinal barrier in C57BL/6 and Ldlr-/- mice and significantly attenuates WD-induced metabolic diseases and, therefore, may represent a novel strategy for management of these diseases.
Assuntos
Aterosclerose/metabolismo , Colesterol na Dieta/administração & dosagem , Dieta Hiperlipídica , Suplementos Nutricionais , Galactose/química , Teste de Tolerância a Glucose , Mucosa Intestinal/metabolismo , Mucinas/metabolismo , Oligossacarídeos/administração & dosagem , Receptores de LDL/genética , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligossacarídeos/químicaRESUMO
In an inflammatory setting, macrophages can be polarized to an inflammatory M1 phenotype or to an anti-inflammatory M2 phenotype, as well as existing on a spectrum between these two extremes. Dysfunction of this phenotypic switch can result in a population imbalance that leads to chronic wounds or disease due to unresolved inflammation. Therapeutic interventions that target macrophages have therefore been proposed and implemented in diseases that feature chronic inflammation such as diabetes mellitus and atherosclerosis. We have developed a model for the sequential influx of immune cells in the peritoneal cavity in response to a bacterial stimulus that includes macrophage polarization, with the simplifying assumption that macrophages can be classified as M1 or M2. With this model, we were able to reproduce the expected timing of sequential influx of immune cells and mediators in a general inflammatory setting. We then fit this model to in vivo experimental data obtained from a mouse peritonitis model of inflammation, which is widely used to evaluate endogenous processes in response to an inflammatory stimulus. Model robustness is explored with local structural and practical identifiability of the proposed model a posteriori. Additionally, we perform sensitivity analysis that identifies the population of apoptotic neutrophils as a key driver of the inflammatory process. Finally, we simulate a selection of proposed therapies including points of intervention in the case of delayed neutrophil apoptosis, which our model predicts will result in a sustained inflammatory response. Our model can therefore provide hypothesis testing for therapeutic interventions that target macrophage phenotype and predict outcomes to be validated by subsequent experimentation.
Assuntos
Inflamação/imunologia , Macrófagos/imunologia , Modelos Imunológicos , Animais , Apoptose/imunologia , Biologia Computacional , Simulação por Computador , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/imunologia , Ativação de Macrófagos , Macrófagos/classificação , Macrófagos Peritoneais/classificação , Macrófagos Peritoneais/imunologia , Camundongos , Neutrófilos/citologia , Neutrófilos/imunologia , FenótipoRESUMO
A causal relationship exists between macrophage cholesterol levels and inflammation, for example, Interleukin-1ß (IL-1ß) secretion. A decrease in intracellular K+ is essential for inflammasome activation/IL-1ß secretion and, herein, we examined the hypothesis that cellular cholesterol affects K+ -channel activity and K+ -efflux using mouse peritoneal macrophages (MPMs) and human/THP1 macrophages. An increase in cellular cholesterol led to a significant increase in K+ currents (> 350% in both MPM and THP1). Enhancing cholesterol efflux returned K+ currents back to basal levels with corresponding increase in intracellular K+ (11.2-14.5%) and reduced IL-1ß secretion (32-62%). These data demonstrate a novel mechanism by which cellular cholesterol modulates inflammation/inflammasome via regulation of K+ -channel activity and intracellular K+ levels. Attenuation of IL-1ß secretion by Nateglinide/Repaglinide further suggests involvement of Kir6 channels.
Assuntos
Exocitose , Interleucina-1beta/metabolismo , Macrófagos Peritoneais/metabolismo , Canais de Potássio/metabolismo , Animais , Células Cultivadas , Colesterol/metabolismo , Humanos , Inflamassomos/metabolismo , Camundongos , Potássio/metabolismo , Células THP-1RESUMO
BACKGROUND: The associated increase in the lipopolysaccharide (LPS) levels and uremic toxins in chronic kidney disease (CKD) has shifted the way we focus on intestinal microbiota. This study shows that a disruption of the intestinal barrier in CKD promotes leakage of LPS from the gut, subsequently decreasing insulin sensitivity. Butyrate treatment improved the intestinal barrier function by increasing colonic mucin and tight junction (TJ) proteins. This modulation further ameliorated metabolic functions such as insulin intolerance and improved renal function. METHODS: Renal failure was induced by 5/6th nephrectomy (Nx) in rats. A group of Nx and control rats received sodium butyrate in drinking water. The Nx groups were compared with sham-operated controls. RESULTS: The Nx rats had significant increases in serum creatinine, urea and proteinuria. These animals had impaired glucose and insulin tolerance and increased gluconeogenesis, which corresponded with decreased glucagon-like peptide-1 (GLP-1) secretion. The Nx animals suffered significant loss of intestinal TJ proteins, colonic mucin and mucin 2 protein. This was associated with a significant increase in circulating LPS, suggesting a leaky gut phenomenon. 5'adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, known to modulate epithelial TJs and glucose metabolism, was significantly reduced in the intestine of the Nx group. Anti-inflammatory cytokine, interleukin 10, anti-bacterial peptide and cathelicidin-related antimicrobial peptide were also lowered in the Nx cohort. Butyrate treatment increased AMPK phosphorylation, improved renal function and controlled hyperglycemia. CONCLUSIONS: Butyrate improves AMPK phosphorylation, increases GLP-1 secretion and promotes colonic mucin and TJ proteins, which strengthen the gut wall. This decreases LPS leakage and inflammation. Taken together, butyrate improves metabolic parameters such as insulin resistance and markers of renal failure in CKD animals.
Assuntos
Ácido Butírico/farmacologia , Resistência à Insulina/fisiologia , Mucosa Intestinal/metabolismo , Mucinas/biossíntese , Insuficiência Renal Crônica/tratamento farmacológico , Animais , Modelos Animais de Doenças , Antagonistas dos Receptores Histamínicos/farmacologia , Imuno-Histoquímica , Masculino , Permeabilidade , Ratos , Insuficiência Renal Crônica/metabolismoRESUMO
Cell membrane-camouflaged nanoparticles have appeared as a promising platform to develop active tumor targeting nanomedicines. To evade the immune surveillance, we designed a composite cell membrane-camouflaged biomimetic nanoplatform, namely, leutusome, which is made of liposomal nanoparticles incorporating plasma membrane components derived from both leukocytes (murine J774A.1 cells) and tumor cells (head and neck tumor cells HN12). Exogenous phospholipids were used as building blocks to fuse with two cell membranes to form liposomal nanoparticles. Liposomal nanoparticles made of exogenous phospholipids only or in combination with one type of cell membrane were fabricated and compared. The anticancer drug paclitaxel (PTX) was used to make drug-encapsulating liposomal nanoparticles. Leutusome resembling characteristic plasma membrane components of the two cell membranes were examined and confirmed in vitro. A xenograft mouse model of head and neck cancer was used to profile the blood clearance kinetics, biodistribution, and antitumor efficacy of the different liposomal nanoparticles. The results demonstrated that leutusome obtained prolonged blood circulation and was most efficient accumulating at the tumor site (79.1 ± 6.6% ID per gram of tumor). Similarly, leutusome composed of membrane fractions of B16 melanoma cells and leukocytes (J774A.1) showed prominent accumulation within the B16 tumor, suggesting the generalization of the approach. Furthermore, PTX-encapsulating leutusome was found to most potently inhibit tumor growth while not causing systemic adverse effects.
Assuntos
Biomimética/métodos , Membrana Celular/metabolismo , Lipossomos/metabolismo , Melanoma Experimental/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Membrana Celular/química , Sistemas de Liberação de Medicamentos , Humanos , Leucócitos , Lipossomos/química , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Nanopartículas/química , Paclitaxel/química , Paclitaxel/farmacologia , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chronic inflammation is a known hallmark of cancer and is central to the onset and progression of hepatocellular carcinoma (HCC). Hepatic macrophages play a critical role in the inflammatory process leading to HCC. The oncogene Astrocyte elevated gene-1 (AEG-1) regulates NFκB activation, and germline knockout of AEG-1 in mice (AEG-1-/-) results in resistance to inflammation and experimental HCC. In this study, we developed conditional hepatocyte- and myeloid cell-specific AEG-1-/- mice (AEG-1ΔHEP and AEG-1ΔMAC, respectively) and induced HCC by treatment with N-nitrosodiethylamine (DEN) and phenobarbital (PB). AEG-1ΔHEP mice exhibited a significant reduction in disease severity compared with control littermates, while AEG-1ΔMAC mice were profoundly resistant. In vitro, AEG-1-/- hepatocytes exhibited increased sensitivity to stress and senescence. Notably, AEG-1-/- macrophages were resistant to either M1 or M2 differentiation with significant inhibition in migration, endothelial adhesion, and efferocytosis activity, indicating that AEG-1 ablation renders macrophages functionally anergic. These results unravel a central role of AEG-1 in regulating macrophage activation and indicate that AEG-1 is required in both tumor cells and tumor microenvironment to stimulate hepatocarcinogenesis.Significance: These findings distinguish a novel role of macrophage-derived oncogene AEG-1 from hepatocellular AEG-1 in promoting inflammation and driving tumorigenesis. Cancer Res; 78(22); 6436-46. ©2018 AACR.
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Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Animais , Adesão Celular , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Quimiotaxia , Dietilnitrosamina , Hepatócitos/citologia , Hepatócitos/metabolismo , Inflamação , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fenobarbital , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Fatores de Risco , Microambiente TumoralRESUMO
Intestinal epithelial cell derived alkaline phosphatase (IAP) dephosphorylates/detoxifies bacterial endotoxin lipopolysaccharide (LPS) in the gut lumen. We have earlier demonstrated that consumption of high-fat high-cholesterol containing western type-diet (WD) significantly reduces IAP activity, increases intestinal permeability leading to increased plasma levels of LPS and glucose intolerance. Furthermore, oral supplementation with curcumin that increased IAP activity improved intestinal barrier function as well as glucose tolerance. To directly test the hypothesis that targeted increase in IAP would protect against WD-induced metabolic consequences, we developed intestine-specific IAP transgenic mice where expression of human chimeric IAP is under the control of intestine-specific villin promoter. This chimeric human IAP contains domains from human IAP and human placental alkaline phosphatase, has a higher turnover number, narrower substrate specificity, and selectivity for bacterial LPS. Chimeric IAP was specifically and uniformly overexpressed in these IAP transgenic (IAPTg) mice along the entire length of the intestine. While IAP activity reduced from proximal P1 segment to distal P9 segment in wild-type (WT) mice, this activity was maintained in the IAPTg mice. Dietary challenge with WD impaired glucose tolerance in WT mice and this intolerance was attenuated in IAPTg mice. Significant decrease in fecal zonulin, a marker for intestinal barrier dysfunction, in WD fed IAPTg mice and a corresponding decrease in translocation of orally administered nonabsorbable 4 kDa FITC dextran to plasma suggests that IAP overexpression improves intestinal barrier function. Thus, targeted increase in IAP activity represents a novel strategy to improve WD-induced intestinal barrier dysfunction and glucose intolerance.
Assuntos
Fosfatase Alcalina/metabolismo , Dieta Ocidental/efeitos adversos , Intolerância à Glucose/metabolismo , Mucosa Intestinal/metabolismo , Fosfatase Alcalina/genética , Animais , Células Cultivadas , Toxina da Cólera/metabolismo , Colesterol/efeitos adversos , Intolerância à Glucose/etiologia , Intolerância à Glucose/genética , Haptoglobinas , Humanos , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Precursores de ProteínasRESUMO
Intracellular cholesterol transport proteins move cholesterol to different subcellular compartments and thereby regulate its final metabolic fate. In hepatocytes, for example, delivery of high-density lipoprotein (HDL)-associated cholesterol for bile acid synthesis or secretion into bile facilitates cholesterol elimination from the body (anti-atherogenic effect), whereas delivery for esterification and subsequent incorporation into apolipoprotein B-containing atherogenic lipoproteins (e.g. very-low-density lipoprotein (VLDL)) enhances cholesterol secretion into the systemic circulation (pro-atherogenic effect). Intracellular cholesterol transport proteins such as sterol carrier protein-2 (SCP2) should, therefore, play a role in regulating these pro- or anti-atherosclerotic processes. Here, we sought to evaluate the effects of SCP2 deficiency on the development of diet-induced atherosclerosis. We generated LDLR-/- mice deficient in SCP2/SCPx (LS) and examined the effects of this deficiency on Western diet-induced atherosclerosis. SCP2/SCPx deficiency attenuated atherosclerosis in LS mice by >80% and significantly reduced plasma cholesterol and triglyceride levels. Investigation of the likely underlying mechanisms revealed a significant reduction in intestinal cholesterol absorption (given as an oral gavage) in SCP2/SCPx-deficient mice. Consistently, siRNA-mediated knockdown of SCP2 in intestinal cells significantly reduced cholesterol uptake. Furthermore, hepatic triglyceride/VLDL secretion from the liver or hepatocytes isolated from SCP2/SCPx-deficient mice was significantly reduced. These results indicate an important regulatory role for SCP2 deficiency in attenuating diet-induced atherosclerosis by limiting intestinal cholesterol absorption and decreasing hepatic triglyceride/VLDL secretion. These findings suggest targeted inhibition of SCP2 as a potential therapeutic strategy to reduce Western diet-induced dyslipidemia and atherosclerosis.
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Aterosclerose/etiologia , Aterosclerose/metabolismo , Proteínas de Transporte/metabolismo , Dieta Ocidental/efeitos adversos , Dislipidemias/etiologia , Dislipidemias/metabolismo , Animais , Aterosclerose/sangue , Aterosclerose/genética , Proteínas de Transporte/genética , Colesterol/sangue , Colesterol/metabolismo , Dislipidemias/sangue , Dislipidemias/genética , Feminino , Deleção de Genes , Absorção Intestinal , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Triglicerídeos/sangue , Triglicerídeos/metabolismoRESUMO
Curcumin has anti-inflammatory, anti-oxidant and anti-proliferative properties established largely by in vitro studies. Accordingly, oral administration of curcumin beneficially modulates many diseases including diabetes, fatty-liver disease, atherosclerosis, arthritis, cancer and neurological disorders such as depression, Alzheimer's or Parkinson's disease. However, limited bioavailability and inability to detect curcumin in circulation or target tissues has hindered the validation of a causal role. We established curcumin-mediated decrease in the release of gut bacteria-derived lipopolysaccharide (LPS) into circulation by maintaining the integrity of the intestinal barrier function as the mechanism underlying the attenuation of metabolic diseases (diabetes, atherosclerosis, kidney disease) by curcumin supplementation precluding the need for curcumin absorption. In view of the causative role of circulating LPS and resulting chronic inflammation in the development of diseases listed above, this review summarizes the mechanism by which curcumin affects the several layers of the intestinal barrier and, despite negligible absorption, can beneficially modulate these diseases.
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Curcumina/uso terapêutico , Intestinos/efeitos dos fármacos , Curcumina/farmacologia , Humanos , Mucosa Intestinal/metabolismoRESUMO
Dysfunctional macrophages underlie the development of several diseases including atherosclerosis where accumulation of cholesteryl esters and persistent inflammation are 2 of the critical macrophage processes that regulate the progression as well as stability of atherosclerotic plaques. Ligand-dependent activation of liver-x-receptor (LXR) not only enhances mobilization of stored cholesteryl ester but also exerts anti-inflammatory effects mediated via trans-repression of proinflammatory transcription factor nuclear factor kappa B. However, increased hepatic lipogenesis by systemic administration of LXR ligands (LXR-L) has precluded their therapeutic use. The objective of the present study was to devise a strategy to selectively deliver LXR-L to atherosclerotic plaque-associated macrophages while limiting hepatic uptake. Mannose-functionalized dendrimeric nanoparticles (mDNP) were synthesized to facilitate active uptake via the mannose receptor expressed exclusively by macrophages using polyamidoamine dendrimer. Terminal amine groups were used to conjugate mannose and LXR-L T091317 via polyethylene glycol spacers. mDNP-LXR-L was effectively taken up by macrophages (and not by hepatocytes), increased expression of LXR target genes (ABCA1/ABCG1), and enhanced cholesterol efflux. When administered intravenously to LDLR-/- mice with established plaques, significant accumulation of fluorescently labeled mDNP-LXR-L was seen in atherosclerotic plaque-associated macrophages. Four weekly injections of mDNP-LXR-L led to significant reduction in atherosclerotic plaque progression, plaque necrosis, and plaque inflammation as assessed by expression of nuclear factor kappa B target gene matrix metalloproteinase 9; no increase in hepatic lipogenic genes or plasma lipids was observed. These studies validate the development of a macrophage-specific delivery platform for the delivery of anti-atherosclerotic agents directly to the plaque-associated macrophages to attenuate plaque burden.
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Aterosclerose/tratamento farmacológico , Dendrímeros/administração & dosagem , Macrófagos/metabolismo , Manose/metabolismo , Nanopartículas/administração & dosagem , Animais , Células Cultivadas , Feminino , Receptores X do Fígado/fisiologia , Masculino , Camundongos , Receptores de LDL/fisiologiaRESUMO
Nonalcoholic steatohepatitis (NASH) is the most prevalent cause of chronic liver disease in the Western world. However, an optimum therapy for NASH is yet to be established, mandating more in-depth investigation into the molecular pathogenesis of NASH to identify novel regulatory molecules and develop targeted therapies. Here, we unravel a unique function of astrocyte elevated gene-1(AEG-1)/metadherin in NASH using a transgenic mouse with hepatocyte-specific overexpression of AEG-1 (Alb/AEG-1) and a conditional hepatocyte-specific AEG-1 knockout mouse (AEG-1ΔHEP ). Alb/AEG-1 mice developed spontaneous NASH whereas AEG-1ΔHEP mice were protected from high-fat diet (HFD)-induced NASH. Intriguingly, AEG-1 overexpression was observed in livers of NASH patients and wild-type (WT) mice that developed steatosis upon feeding HFD. In-depth molecular analysis unraveled that inhibition of peroxisome proliferator-activated receptor alpha activity resulting in decreased fatty acid ß-oxidation, augmentation of translation of fatty acid synthase resulting in de novo lipogenesis, and increased nuclear factor kappa B-mediated inflammation act in concert to mediate AEG-1-induced NASH. Therapeutically, hepatocyte-specific nanoparticle-delivered AEG-1 small interfering RNA provided marked protection from HFD-induced NASH in WT mice. CONCLUSION: AEG-1 might be a key molecule regulating initiation and progression of NASH. AEG-1 inhibitory strategies might be developed as a potential therapeutic intervention in NASH patients. (Hepatology 2017;66:466-480).