RESUMO
Although epidemiological data and results from rodent studies support an inverse relationship between nicotine consumption and body weight, the molecular mechanisms are poorly understood. CD-1 mice were fed a basal diet or a basal diet containing low or high dose smokeless tobacco blend or high dose nicotine tartrate for 14 weeks. High dose tobacco blend and nicotine tartrate diets vs. basal diet reduced mouse body weight (16.3% and 19.7%, respectively), epididymal (67.6% and 72.5%, respectively) and brown adipose weight (42% and 38%, respectively), epididymal adipocyte size (46.4% and 41.4%, respectively), and brown adipose tissue lipid droplet abundance, with no elevation of adipose tissue inflammation. High dose tobacco blend and nicotine diets also increased mouse physical activity and decreased respiratory exchange ratio, suggesting that high dose nicotine intake induces adipose tissue triglyceride lipolysis to provide fatty acids as an energy source. Both low and high dose tobacco blend and nicotine diet feeding vs. basal diet increased plasma insulin levels (2.9, 3.6 and 4.3-fold, respectively) and improved blood glucose disposal without affecting insulin sensitivity. Feeding of the high dose tobacco blend or nicotine feeding in mice induces body weight loss likely by increasing physical activity and stimulating adipose tissue triglyceride lipolysis.
Assuntos
Adipócitos/efeitos dos fármacos , Nicotiana , Nicotina/farmacologia , Condicionamento Físico Animal , Redução de Peso/efeitos dos fármacos , Adipócitos/citologia , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Tamanho Celular , Comportamento Alimentar , Insulina/sangue , Resistência à Insulina , Lipólise , Masculino , Camundongos , Triglicerídeos/metabolismoRESUMO
Apolipoprotein M (apoM), a plasma sphingosine 1-phosphate (S1P) carrier, associates with plasma HDL via its uncleaved signal peptide. Hepatocyte-specific apoM overexpression in mice stimulates formation of both larger nascent HDL in hepatocytes and larger mature apoM/S1P-enriched HDL particles in plasma by enhancing hepatic S1P synthesis and secretion. Mutagenesis of apoM glutamine 22 to alanine (apoM(Q22A)) introduces a functional signal peptidase cleavage site. Expression of apoM(Q22A) in ABCA1-expressing HEK293 cells resulted in the formation of smaller nascent HDL particles compared with wild type apoM (apoM(WT)). When apoM(Q22A) was expressed in vivo, using recombinant adenoviruses, smaller plasma HDL particles and decreased plasma S1P and apoM were observed relative to expression of apoM(WT). Hepatocytes isolated from both apoM(WT)- and apoM(Q22A)-expressing mice displayed an equivalent increase in cellular levels of S1P, relative to LacZ controls; however, relative to apoM(WT), apoM(Q22A) hepatocytes displayed more rapid apoM and S1P secretion but minimal apoM(Q22A) bound to nascent lipoproteins. Pharmacologic inhibition of ceramide synthesis increased cellular sphingosine and S1P but not medium S1P in both apoM(WT) and apoM(Q22A) hepatocytes. We conclude that apoM secretion is rate-limiting for hepatocyte S1P secretion and that its uncleaved signal peptide delays apoM trafficking out of the cell, promoting formation of larger nascent apoM- and S1P-enriched HDL particles that are probably precursors of larger apoM/S1P-enriched plasma HDL.
Assuntos
Apolipoproteínas/metabolismo , Lipoproteínas HDL/química , Lisofosfolipídeos/química , Sinais Direcionadores de Proteínas , Esfingosina/análogos & derivados , Animais , Apolipoproteínas/química , Apolipoproteínas M , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Esfingosina/químicaRESUMO
OBJECTIVE: Transplantation studies suggest that bone marrow cell ATP-binding cassette transporter A1 protects against atherosclerosis development. However, the in vivo effect of macrophage ATP-binding cassette transporter A1 expression on atherogenesis is not fully understood because bone marrow contains other leukocytes and hematopoietic stem and progenitor cells. Myeloid-specific ATP-binding cassette transporter A1 knockout mice in the low-density lipoprotein (LDL) receptor knockout C57BL/6 background were developed to address this question. APPROACH AND RESULTS: Chow-fed myeloid-specific ATP-binding cassette transporter A1 knockout/LDL receptor knockout (double knockout [DKO]) versus LDL receptor knockout (single knockout [SKO]) mice had similar plasma lipid concentrations, but atherogenic diet (AD)-fed DKO mice had reduced plasma very-LDL (VLDL)/LDL concentrations resulting from decreased hepatic VLDL triglyceride secretion. Resident peritoneal macrophages from AD-fed DKO versus SKO mice had significantly higher cholesterol content but similar proinflammatory gene expression. Atherosclerosis extent was similar between genotypes after 10 to 16 weeks of AD but increased modestly in DKO mice by 24 weeks of AD. Lesional macrophage content was similar, likely because of the higher monocyte flux through aortic root lesions in DKO versus SKO mice. After transplantation of DKO or SKO bone marrow into SKO mice and 16 weeks of AD feeding, atherosclerosis extent was similar and plasma apolipoprotein B lipoproteins were reduced in mice receiving DKO bone marrow. When differences in plasma VLDL/LDL concentrations were minimized by maintaining mice on chow for 24 weeks, DKO mice had modest, but significantly more, atherosclerosis compared with SKO mice. CONCLUSIONS: Myeloid cell ATP-binding cassette transporter A1 increases hepatic VLDL triglyceride secretion and plasma VLDL/LDL concentrations in AD-fed LDL receptor knockout mice, offsetting its atheroprotective role in decreasing macrophage cholesterol content, resulting in a minimal increase in atherosclerosis.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/fisiologia , Aterosclerose/metabolismo , Colesterol/metabolismo , Dieta Aterogênica/efeitos adversos , Macrófagos Peritoneais/metabolismo , Células Mieloides/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/genética , Aterosclerose/prevenção & controle , Transplante de Medula Óssea , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Feminino , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/deficiência , Receptores de LDL/genética , Triglicerídeos/sangueRESUMO
Apolipoprotein M (apoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a signaling molecule active in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulated larger nascent HDL formation, compared with cells that did not express apoM; however, the in vivo role of apoM in HDL metabolism remains poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated hepatocyte-specific apoM transgenic (APOM Tg) mice, which had an â¼3-5-fold increase in plasma apoM levels compared with wild-type mice. Although HDL cholesterol concentrations were similar to wild-type mice, APOM Tg mice had larger plasma HDLs enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase, and S1P. Despite the presence of larger plasma HDLs in APOM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to that in wild-type mice. APOM Tg mice had an â¼5-fold increase in plasma S1P, which was predominantly associated with larger plasma HDLs. Primary hepatocytes from APOM Tg mice generated larger nascent HDLs and displayed increased sphingolipid synthesis and S1P secretion. Inhibition of ceramide synthases in hepatocytes increased cellular S1P levels but not S1P secretion, suggesting that apoM is rate-limiting in the export of hepatocyte S1P. Our data indicate that hepatocyte-specific apoM overexpression generates larger nascent HDLs and larger plasma HDLs, which preferentially bind apoM and S1P, and stimulates S1P biosynthesis for secretion. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection and may have other as yet unidentified functions.
Assuntos
Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Hepatócitos/metabolismo , Lipocalinas/genética , Lipocalinas/metabolismo , Lipoproteínas HDL/metabolismo , Fígado/metabolismo , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Animais , Apolipoproteínas E/sangue , Apolipoproteínas M , Feminino , Humanos , Lipoproteínas HDL/sangue , Lipoproteínas HDL/química , Fígado/citologia , Lisofosfolipídeos/biossíntese , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Tamanho da Partícula , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Esfingosina/biossíntese , Esfingosina/metabolismoRESUMO
Echium oil (EO), which is enriched in SDA (18:4 n-3), reduces plasma triglyceride (TG) concentrations in humans and mice. We compared mechanisms by which EO and fish oil (FO) reduce plasma TG concentrations in mildly hypertriglyceridemic male apoB100-only LDLrKO mice. Mice were fed one of three atherogenic diets containing 0.2% cholesterol and palm oil (PO; 20%), EO (10% EO + 10% PO), or FO (10% FO + 10% PO). Livers from PO- and EO-fed mice had similar TG and cholesteryl ester (CE) content, which was significantly higher than in FO-fed mice. Plasma TG secretion was reduced in FO vs. EO-fed mice. Plasma very low density lipoprotein (VLDL) particle size was ordered: PO (63 ± 4 nm) > EO (55 ± 3 nm) > FO (40 ± 2 nm). Post-heparin lipolytic activity was similar among groups, but TG hydrolysis by purified lipoprotein lipase was significantly greater for EO and FO VLDL compared to PO VLDL. Removal of VLDL tracer from plasma was marginally faster in EO vs. PO fed mice. Our results suggest that EO reduces plasma TG primarily through increased intravascular lipolysis of TG and VLDL clearance. Finally, EO may substitute for FO to reduce plasma TG concentrations, but not hepatic steatosis in this mouse model.
Assuntos
Apolipoproteína B-100/sangue , Echium/química , Lipólise/efeitos dos fármacos , Óleos de Plantas/farmacologia , Receptores de LDL/sangue , Triglicerídeos/sangue , Animais , Ésteres do Colesterol/sangue , VLDL-Colesterol/sangue , Óleos de Peixe/farmacologia , Expressão Gênica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óleo de Palmeira , RNA/genética , RNA/isolamento & purificaçãoRESUMO
Obesity-associated low-grade chronic inflammation plays an important role in the development of insulin resistance. The membrane lipid transporter ATP-binding cassette transporter A1 (ABCA1) promotes formation of nascent HDL particles. ABCA1 also dampens macrophage inflammation by reducing cellular membrane cholesterol and lipid raft content. We tested the hypothesis that myeloid-specific ABCA1 deletion may exacerbate insulin resistance by increasing the obesity-associated chronic low-grade inflammation. Myeloid cell-specific ABCA1 knockout (MSKO) and wild-type (WT) mice developed obesity, insulin resistance, mild hypercholesterolemia, and hepatic steatosis to a similar extent with a 45% high-fat (HF) diet feeding or after crossing into the ob/ob background. Resident peritoneal macrophages and stromal vascular cells from obese MSKO mice accumulated significantly more cholesterol. Relative to chow, HF diet markedly induced macrophage infiltration and inflammatory cytokine expression to a similar extent in adipose tissue of WT and MSKO mice. Among pro-inflammatory cytokines examined, only IL-6 was highly upregulated in MSKO-ob/ob versus ob/ob mouse peritoneal macrophages, indicating a nonsignificant effect of myeloid ABCA1 deficiency on obesity-associated chronic inflammation. In conclusion, myeloid-specific ABCA1 deficiency does not exacerbate obesity-associated low-grade chronic inflammation and has minimal impact on the pathogenesis of insulin resistance in both HF diet-induced and genetically obese mouse models.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/genética , Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina , Macrófagos/metabolismo , Obesidade/metabolismo , Transportador 1 de Cassete de Ligação de ATP/deficiência , Tecido Adiposo Branco/imunologia , Tecido Adiposo Branco/metabolismo , Animais , Células Cultivadas , Quimiocinas/metabolismo , Colesterol/sangue , Epididimo/imunologia , Epididimo/metabolismo , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Camundongos Transgênicos , Obesidade/etiologia , Obesidade/imunologiaRESUMO
RATIONALE: ATP-binding cassette transporter A1 (ABCA1) plays a critical role in eliminating excess free cholesterol from tissues by effluxing cellular free cholesterol and phospholipids to lipid-poor apolipoprotein AI. Macrophage ABCA1 also dampens proinflammatory myeloid differentiation primary-response protein 88-dependent toll-like receptor signaling by reducing cellular membrane free cholesterol and lipid raft content, indicating a role of ABCA1 in innate immunity. However, whether ABCA1 expression has a role in regulating macrophage function in vivo is unknown. OBJECTIVE: We investigated whether macrophage ABCA1 expression impacts host defense function, including microbial killing and chemotaxis. METHODS AND RESULTS: Myeloid cell-specific ABCA1 knockout (MSKO) vs wild-type mice were infected with Listeria monocytogenes (Lm) for 36 hours or 72 hours before euthanasia. Lm-induced monocytosis was similar for wild-type and MSKO mice; however, MSKO mice were more resistant to Lm infection, with significantly less body weight loss, less Lm burden in liver and spleen, and less hepatic damage 3 days postinfection. In addition, Lm-infected MSKO mouse livers had: (1) greater monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 expression; (2) more monocyte/macrophage infiltration; (3) less neutral lipid accumulation; and (4) diminished expression of lipogenic genes. MSKO macrophages showed enhanced chemotaxis toward chemokines in vitro and increased migration from peritoneum in response to lipopolysaccharide in vivo. Lm infection of wild-type macrophages markedly reduced expression of ABCA1 protein, as well as other cholesterol export proteins (such as ATP-binding cassette transporter G1 and apolipoprotein E). CONCLUSIONS: Myeloid-specific ABCA1 deletion favors host response to and clearance of Lm. Macrophage Lm infection reduces expression of cholesterol export proteins, suggesting that diminished cholesterol efflux enhances innate immune function of macrophages.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Células Mieloides/imunologia , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/imunologia , Apolipoproteínas E/metabolismo , Apoptose/imunologia , Western Blotting , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Células Cultivadas , Quimiocinas/sangue , Quimiocinas/genética , Quimiocinas/imunologia , Citocinas/sangue , Citocinas/genética , Citocinas/imunologia , Resistência à Doença/genética , Resistência à Doença/imunologia , Feminino , Interações Hospedeiro-Patógeno/imunologia , Listeria monocytogenes/fisiologia , Listeriose/genética , Listeriose/microbiologia , Fígado/imunologia , Fígado/microbiologia , Fígado/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/patologia , Células Mieloides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: Fish oil, containing omega-3 fatty acids, attenuates atherosclerosis. We hypothesized that omega-3 fatty acid-enriched oils are atheroprotective through alteration of monocyte subsets and their trafficking into atherosclerotic lesions. METHODS AND RESULTS: Low-density lipoprotein receptor knockout and apolipoprotein E(-/-) mice were fed diets containing 10% (calories) palm oil and 0.2% cholesterol, supplemented with an additional 10% palm oil, echium oil (containing 18:4 n-3), or fish oil. Compared with palm oil-fed low-density lipoprotein receptor knockout mice, echium oil and fish oil significantly reduced plasma cholesterol, splenic Ly6C(hi) monocytosis by ≈50%, atherosclerosis by 40% to 70%, monocyte trafficking into the aortic root by ≈50%, and atherosclerotic lesion macrophage content by 30% to 44%. In contrast, atherosclerosis and monocyte trafficking into the artery wall was not altered by omega-3 fatty acids in apolipoprotein E(-/-) mice; however, Ly6C(hi) splenic monocytes positively correlated with aortic root intimal area across all diet groups. In apolipoprotein E(-/-) mice, fish oil reduced the percentage of blood Ly6C(hi) monocytes, despite an average 2-fold higher plasma cholesterol relative to palm oil. CONCLUSIONS: The presence of splenic Ly6C(hi) monocytes parallels the appearance of atherosclerotic disease in both low-density lipoprotein receptor knockout and apolipoprotein E(-/-) mice. Furthermore, omega-3 fatty acids favorably alter monocyte subsets independently from effects on plasma cholesterol and reduce monocyte recruitment into atherosclerotic lesions.
Assuntos
Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Quimiotaxia/efeitos dos fármacos , Suplementos Nutricionais , Ácidos Graxos Ômega-3/farmacologia , Monócitos/efeitos dos fármacos , Óleos de Plantas/farmacologia , Baço/efeitos dos fármacos , Animais , Antígenos Ly/sangue , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/sangue , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Biomarcadores/sangue , Colesterol na Dieta/sangue , Modelos Animais de Doenças , Echium , Feminino , Mediadores da Inflamação/sangue , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/patologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Óleo de Palmeira , Receptores de LDL/deficiência , Receptores de LDL/genética , Baço/imunologia , Baço/metabolismo , Fatores de TempoRESUMO
12/15 lipoxygenase (12/15LO) oxidizes polyunsaturated fatty acids (PUFAs) to form bioactive lipid mediators. The role of 12/15LO in atherosclerosis development remains controversial. We evaluated atherosclerosis development and lipid metabolism in 12/15LO-LDL receptor (LDLr) double knockout (DK) vs. LDLr knockout (SK) mice fed a PUFA-enriched diet to enhance production of 12/15LO products. Compared with SK controls, DK mice fed a PUFA-enriched diet had decreased plasma and liver lipid levels, hepatic lipogenic gene expression, VLDL secretion, and aortic atherosclerosis and increased VLDL turnover. Bone marrow transplantation and Kupffer cell ablation studies suggested both circulating leukocytes and Kupffer cells contributed to the lipid phenotype in 12/15LO-deficient mice. Conditioned medium from in vitro incubation of DK vs. SK macrophages reduced triglyceride secretion in McArdle 7777 hepatoma cells. Our results suggest that, in the context of dietary PUFA enrichment, macrophage 12/15LO expression adversely affects plasma and hepatic lipid metabolism, resulting in exacerbated atherosclerosis.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Aterosclerose/patologia , Metabolismo dos Lipídeos , Macrófagos/enzimologia , Animais , Transplante de Medula Óssea , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Meios de Cultivo Condicionados , Dieta Aterogênica/efeitos adversos , Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Graxos Insaturados/administração & dosagem , Ácidos Graxos Insaturados/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Hepatócitos/metabolismo , Células de Kupffer/metabolismo , Leucócitos/metabolismo , Lipoproteínas VLDL/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Placa Aterosclerótica/patologiaRESUMO
In Tangier disease, absence of ATP binding cassette transporter A1 (ABCA1) results in reduced plasma HDL and elevated triglyceride (TG) levels. We hypothesized that hepatocyte ABCA1 regulates VLDL TG secretion through nascent HDL production. Silencing of ABCA1 expression in oleate-stimulated rat hepatoma cells resulted in: 1) decreased large nascent HDL (>10 nm diameter) and increased small nascent HDL (<10 nm) formation, 2) increased large buoyant VLDL1 particle secretion, and 3) decreased phosphatidylinositol-3 (PI3) kinase activation. Nascent HDL-containing conditioned medium from rat hepatoma cells or HEK293 cells transfected with ABCA1 was effective in increasing PI3 kinase activation and reducing VLDL TG secretion in ABCA1-silenced hepatoma cells. Addition of isolated large nascent HDL particles to ABCA1-silenced hepatoma cells inhibited VLDL TG secretion to a greater extent than small nascent HDL. Similarly, addition of recombinant HDL, but not human plasma HDL, was effective in attenuating TG secretion and increasing PI3 kinase activation in ABCA1-silenced cells. Collectively, these data suggest that large nascent HDL particles, assembled by hepatic ABCA1, generate a PI3 kinase-mediated autocrine signal that attenuates VLDL maturation and TG secretion. This pathway may explain the elevated plasma TG concentration that occurs in most Tangier subjects and may also account, in part, for the inverse relationship between plasma HDL and TG concentrations in individuals with compromised ABCA1 function.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Lipoproteínas de Alta Densidade Pré-beta/fisiologia , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Triglicerídeos/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apolipoproteína A-I/análise , Apolipoproteína A-I/metabolismo , Apolipoproteínas B/análise , Eletroforese das Proteínas Sanguíneas , Linhagem Celular , Linhagem Celular Tumoral , Centrifugação com Gradiente de Concentração , Ativação Enzimática , Humanos , Lipoproteínas HDL/química , Lipoproteínas HDL/isolamento & purificação , Lipoproteínas HDL/fisiologia , Lipoproteínas VLDL/química , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Ratos , Doença de Tangier/fisiopatologiaRESUMO
OBJECTIVE: Activated endothelium and increased monocyte-endothelial interactions in the vessel wall are key early events in atherogenesis. ATP binding cassette (ABC) transporters play important roles in regulating sterol homeostasis in many cell types. Endothelial cells (EC) have a high capacity to efflux sterols and express the ABC transporter, ABCG1. Here, we define the role of ABCG1 in the regulation of lipid homeostasis and inflammation in aortic EC. METHODS AND RESULTS: Using EC isolated from ABCG1-deficient mice (ABCG1 KO), we observed reduced cholesterol efflux to high-density lipoprotein compared to C57BL/6 (B6) EC. However, total cholesteryl ester levels were not changed in ABCG1 KO EC. Secretions of KC, MCP-1, and IL-6 by ABCG1 KO EC were significantly increased, and surface expressions of intercellular adhesion molecule-1 and E-selectin were increased several-fold on ABCG1 KO EC. Concomitant with these findings, we observed a 4-fold increase in monocyte adhesion to the intact aortic endothelium of ABCG1 KO mice ex vivo and to isolated aortic EC from these mice in vitro. In a gain-of-function study in vitro, restoration of ABCG1 expression in ABCG1 KO EC reduced monocyte-endothelial interactions. Utilizing pharmacological inhibitors for STAT3 and the IL-6 receptor, we found that blockade of STAT3 and IL-6 receptor signaling in ABCG1 KO EC completely abrogated monocyte adhesion to ABCG1 KO endothelium. CONCLUSIONS: ABCG1 deficiency in aortic endothelial cells activates endothelial IL-6-IL-6 receptor-STAT3 signaling, thereby increasing monocyte-endothelial interactions and vascular inflammation.
Assuntos
Adesão Celular , Células Endoteliais/metabolismo , Inflamação/metabolismo , Lipoproteínas/deficiência , Monócitos/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Quimiotaxia , Colesterol/metabolismo , HDL-Colesterol/metabolismo , Selectina E/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Lipoproteínas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We investigated the in vivo metabolic fate of pre-beta HDL particles in human apolipoprotein A-I transgenic (hA-I (Tg)) mice. Pre-beta HDL tracers were assembled by incubation of [(125)I]tyramine cellobiose-labeled apolipoprotein A-I (apoA-I) with HEK293 cells expressing ABCA1. Radiolabeled pre-beta HDLs of increasing size (pre-beta1, -2, -3, and -4 HDLs) were isolated by fast-protein liquid chromatography and injected into hA-I (Tg)-recipient mice, after which plasma decay, in vivo remodeling, and tissue uptake were monitored. Pre-beta2, -3, and -4 had similar plasma die-away rates, whereas pre-beta1 HDL was removed 7-fold more rapidly. Radiolabel recovered in liver and kidney 24 h after tracer injection suggested increased (P < 0.001) liver and decreased kidney catabolism as pre-beta HDL size increased. In plasma, pre-beta1 and -2 were rapidly (<5 min) remodeled into larger HDLs, whereas pre-beta3 and -4 were remodeled into smaller HDLs. Pre-beta HDLs were similarly remodeled in vitro with control or LCAT-immunodepleted plasma, but not when incubated with phospholipid transfer protein knockout plasma. Our results suggest that initial interaction of apoA-I with ABCA1 imparts a unique conformation that partially determines the in vivo metabolic fate of apoA-I, resulting in increased liver and decreased kidney catabolism as pre-beta HDL particle size increases.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , Lipoproteínas de Alta Densidade Pré-beta/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Conformação Proteica , RatosRESUMO
Macrophage-specific Abca1 knock-out (Abca1(-)(M)(/-)(M)) mice were generated to determine the role of macrophage ABCA1 expression in plasma lipoprotein concentrations and the innate immune response of macrophages. Plasma lipid and lipoprotein concentrations in chow-fed Abca1(-)(M)(/-)(M) and wild-type (WT) mice were indistinguishable. Compared with WT macrophages, Abca1(-)(M)(/-)(M) macrophages had a >95% reduction in ABCA1 protein, failed to efflux lipid to apoA-I, and had a significant increase in free cholesterol (FC) and membrane lipid rafts without induction of endoplasmic reticulum stress. Lipopolysaccharide (LPS)-treated Abca1(-)(M)(/-)(M) macrophages exhibited enhanced expression of pro-inflammatory cytokines and increased activation of the NF-kappaB and MAPK pathways, which could be diminished by silencing MyD88 or by chemical inhibition of NF-kappaB or MAPK. In vivo LPS injection also resulted in a higher pro-inflammatory response in Abca1(-)(M)(/-)(M) mice compared with WT mice. Furthermore, cholesterol depletion of macrophages with methyl-beta-cyclodextrin normalized FC content between the two genotypes and their response to LPS; cholesterol repletion of macrophages resulted in increased cellular FC accumulation and enhanced cellular response to LPS. Our results suggest that macrophage ABCA1 expression may protect against atherosclerosis by facilitating the net removal of excess lipid from macrophages and dampening pro-inflammatory MyD88-dependent signaling pathways by reduction of cell membrane FC and lipid raft content.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Colesterol/metabolismo , Macrófagos/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/fisiologia , Animais , Inflamação , Sistema de Sinalização das MAP Quinases , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fator 88 de Diferenciação Mieloide/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais , beta-Ciclodextrinas/metabolismoRESUMO
We tested the hypothesis that dietary supplementation with echium oil (EO), which is enriched in stearidonic acid (SDA; 18:4 n-3), the product of Delta-6 desaturation of 18:3 n-3, will decrease plasma triglyceride (TG) concentrations and result in conversion of SDA to eicosapentaenoic acid (EPA) in the liver. Mildly hypertriglyceridemic mice (apoB100-only LDLrKO) were fed a basal diet containing 10% calories as palm oil (PO) and 0.2% cholesterol for 4 weeks, after which they were randomly assigned to experimental diets consisting of the basal diet plus supplementation of 10% of calories as PO, EO or fish oil (FO) for 8 weeks. The EO and FO experimental diets decreased plasma TG and VLDL lipid concentration, and hepatic TG content compared to PO, and there was a significant correlation between hepatic TG content and plasma TG concentration among diet groups. EO fed mice had plasma and liver lipid EPA enrichment that was greater than PO-fed mice but less than FO-fed mice. Down-regulation of several genes involved in hepatic TG biosynthesis was similar for mice fed EO and FO and significantly lower compared to those fed PO. In conclusion, EO may provide a botanical alternative to FO for reduction of plasma TG concentrations.