RESUMO
apoM is a minor HDL apolipoprotein and carrier for sphingosine-1-phosphate (S1P). HDL apoM and S1P concentrations are inversely associated with atherosclerosis progression in rodents. We evaluated associations between plasma concentrations of S1P, plasma concentrations of apoM, and HDL apoM levels with prevalent subclinical atherosclerosis and mortality in the African American-Diabetes Heart Study participants (N = 545). Associations between plasma S1P, plasma apoM, and HDL apoM with subclinical atherosclerosis and mortality were assessed using multivariate parametric, nonparametric, and Cox proportional hazards models. At baseline, participants' median (25th percentile, 75th percentile) age was 55 (49, 62) years old and their coronary artery calcium (CAC) mass score was 26.5 (0.0, 346.5). Plasma S1P, plasma apoM, and HDL apoM were not associated with CAC. After 64 (57.6, 70.3) months of follow-up, 81 deaths were recorded. Higher concentrations of plasma S1P [odds ratio (OR) = 0.14, P = 0.01] and plasma apoM (OR = 0.10, P = 0.02), but not HDL apoM (P = 0.89), were associated with lower mortality after adjusting for age, sex, statin use, CAC, kidney function, and albuminuria. We conclude that plasma S1P and apoM concentrations are inversely and independently associated with mortality, but not CAC, in African Americans with type 2 diabetes after accounting for conventional risk factors.
Assuntos
Apolipoproteínas M/sangue , Negro ou Afro-Americano , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/mortalidade , Lisofosfolipídeos/sangue , Esfingosina/análogos & derivados , Biomarcadores/sangue , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esfingosina/sangue , Taxa de SobrevidaRESUMO
Type 2 diabetes is a complex disorder affected by multiple genes and the environment. Our laboratory has shown that in response to a glucose challenge, two-pore channel 2 ( Tpcn2) knockout mice exhibit a decreased insulin response but normal glucose clearance, suggesting they have improved insulin sensitivity compared with wild-type mice. We tested the hypothesis that improved insulin sensitivity in Tpcn2 knockout mice would protect against the negative effects of a high fat diet. Male and female Tpcn2 knockout (KO), heterozygous (Het), and wild-type (WT) mice were fed a low-fat (LF) or high-fat (HF) diet for 24 wk. HF diet significantly increases body weight in WT mice relative to those on the LF diet; this HF diet-induced increase in body weight is blunted in the Het and KO mice. Despite the protection against diet-induced weight gain, however, Tpcn2 KO mice are not protected against HF-diet-induced changes in glucose or insulin area under the curve during glucose tolerance tests in female mice, while HF diet has no significant effect on glucose tolerance in the male mice, regardless of genotype. Glucose disappearance during an insulin tolerance test is augmented in male KO mice, consistent with our previous findings suggesting enhanced insulin sensitivity in these mice. Male KO mice exhibit increased fasting plasma total cholesterol and triglyceride concentrations relative to WT mice on the LF diet, but this difference disappears in HF diet-fed mice where there is increased cholesterol and triglycerides across all genotypes. These data demonstrate that knockout of Tpcn2 may increase insulin action in male, but not female, mice. In addition, both male and female KO mice are protected against diet-induced weight gain, but this protection is likely independent from glucose tolerance, insulin sensitivity, and plasma lipid levels.
Assuntos
Canais de Cálcio/genética , Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina/genética , Obesidade/genética , Aumento de Peso/genética , Animais , Canais de Cálcio/metabolismo , Colesterol/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Teste de Tolerância a Glucose , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/metabolismo , Fatores Sexuais , Triglicerídeos/sangueRESUMO
Acquisition of a 'quiescence programme' by naive T cells is important to provide a stress-free environment and resistance to apoptosis while preserving their responsiveness to activating stimuli. Therefore, the survival and proper function of naive T cells depends on their ability to maintain quiescence. Recently we demonstrated that by preventing chronic unresolved endoplasmic reticulum (ER) stress, Schlafen2 (Slfn2) maintains a stress-free environment to conserve a pool of naive T cells ready to respond to a microbial invasion. These findings strongly suggest an intimate association between quiescence and stress signalling. However, the connection between ER stress conditions and loss of T-cell quiescence is unknown. Here we demonstrate that homeostasis of cholesterol and lipids, is disrupted in T cells and monocytes from Slfn2-mutant, elektra, mice with higher levels of lipid rafts and lipid droplets found in these cells. Moreover, elektra T cells had elevated levels of free cholesterol and cholesteryl ester due to increased de novo synthesis and higher levels of the enzyme HMG-CoA reductase. As cholesterol plays an important role in the transition of T cells from resting to active state, and ER regulates cholesterol and lipid synthesis, we suggest that regulation of cholesterol levels through the prevention of ER stress is an essential component of the mechanism by which Slfn2 regulates quiescence.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Senescência Celular , Colesterol/biossíntese , Ativação Linfocitária , Mutação , Linfócitos T/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Ésteres do Colesterol/biossíntese , Estresse do Retículo Endoplasmático , Genótipo , Hidroximetilglutaril-CoA Redutases/metabolismo , Gotículas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Monócitos/imunologia , Monócitos/metabolismo , Fenótipo , Linfócitos T/imunologia , Regulação para CimaRESUMO
APOL1 gene renal-risk variants are associated with nephropathy and CVD in African Americans; however, little is known about the circulating APOL1 variant proteins which reportedly bind to HDL. We examined whether APOL1 G1 and G2 renal-risk variant serum concentrations or lipoprotein distributions differed from nonrisk G0 APOL1 in African Americans without nephropathy. Serum APOL1 protein concentrations were similar regardless of APOL1 genotype. In addition, serum APOL1 protein was bound to protein complexes in two nonoverlapping peaks, herein referred to as APOL1 complex A (12.2 nm diameter) and complex B (20.0 nm diameter). Neither of these protein complexes associated with HDL or LDL. Proteomic analysis revealed that complex A was composed of APOA1, haptoglobin-related protein (HPR), and complement C3, whereas complex B contained APOA1, HPR, IgM, and fibronectin. Serum HPR was less abundant on complex B in individuals with G1 and G2 renal-risk variant genotypes, relative to G0 (P = 0.0002-0.037). These circulating complexes may play roles in HDL metabolism and susceptibility to CVD.
Assuntos
Apolipoproteínas/sangue , Negro ou Afro-Americano , Lipoproteínas HDL/sangue , Adulto , Apolipoproteína L1 , Apolipoproteínas/genética , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Humanos , Nefropatias/sangue , Nefropatias/genética , Lipoproteínas HDL/genética , Masculino , Pessoa de Meia-Idade , Proteômica , Fatores de RiscoRESUMO
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
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
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
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: Hepatic ATP binding cassette transporter A1 (ABCA1) expression is critical for maintaining plasma high-density lipoprotein (HDL) concentrations, but its role in macrophage reverse cholesterol transport and atherosclerosis is not fully understood. We investigated atherosclerosis development and reverse cholesterol transport in hepatocyte-specific ABCA1 knockout (HSKO) mice in the low-density lipoprotein (LDL) receptor KO (LDLrKO) C57BL/6 background. APPROACH AND RESULTS: Male and female LDLrKO and HSKO/LDLrKO mice were switched from chow at 8 weeks of age to an atherogenic diet (10% palm oil, 0.2% cholesterol) for 16 weeks. Chow-fed HSKO/LDLrKO mice had HDL concentrations 10% to 20% of LDLrKO mice, but similar very low-density lipoprotein and LDL concentrations. Surprisingly, HSKO/LDLrKO mice fed the atherogenic diet had significantly lower (40% to 60%) very low-density lipoprotein, LDL, and HDL concentrations (50%) compared with LDLrKO mice. Aortic surface lesion area and cholesterol content were similar for both genotypes of mice, but aortic root intimal area was significantly lower (20% to 40%) in HSKO/LDLrKO mice. Although macrophage (3)H-cholesterol efflux to apoB lipoprotein-depleted plasma was 24% lower for atherogenic diet-fed HSKO/LDLrKO versus LDLrKO mice, variation in percentage efflux among individual mice was <2-fold compared with a 10-fold variation in plasma HDL concentrations, suggesting that HDL levels, per se, were not the primary determinant of plasma efflux capacity. In vivo reverse cholesterol transport, resident peritoneal macrophage sterol content, biliary lipid composition, and fecal cholesterol mass were similar between both genotypes of mice. CONCLUSIONS: The markedly reduced plasma HDL pool in HSKO/LDLrKO mice is sufficient to maintain macrophage reverse cholesterol transport, which, along with reduced plasma very low-density lipoprotein and LDL concentrations, prevented the expected increase in atherosclerosis.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/deficiência , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Colesterol/metabolismo , Fígado/metabolismo , Macrófagos Peritoneais/metabolismo , Receptores de LDL/deficiência , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Doenças da Aorta/etiologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Apolipoproteína B-100 , Apolipoproteínas B/sangue , Aterosclerose/etiologia , Aterosclerose/genética , Aterosclerose/patologia , Bile/metabolismo , Transporte Biológico , Linhagem Celular , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Dieta Aterogênica , Modelos Animais de Doenças , Fezes/química , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/genética , Fatores de TempoRESUMO
ATP-binding cassette transporter G1 (ABCG1) plays a role in the intracellular transport of cholesterol. Invariant NKT (iNKT) cells are a subpopulation of T lymphocytes that recognize glycolipid Ags. In this study, we demonstrate that ABCG1 regulates iNKT cell development and functions in a cell-intrinsic manner. Abcg1(-/-) mice displayed reduced frequencies of iNKT cells in thymus and periphery. Thymic iNKT cells deficient in ABCG1 had reduced membrane lipid raft content, and showed impaired proliferation and defective maturation during the early stages of development. Moreover, we found that Abcg1(-/-) mice possess a higher frequency of Vß7(+) iNKT cells, suggesting alterations in iNKT cell thymic selection. Furthermore, in response to CD3ε/CD28 stimulation, Abcg1(-/-) thymic iNKT cells showed reduced production of IL-4 but increased production of IFN-γ. Our results demonstrate that changes in intracellular cholesterol homeostasis by ABCG1 profoundly impact iNKT cell development and function.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Colesterol/imunologia , Regulação da Expressão Gênica/imunologia , Lipoproteínas/imunologia , Células T Matadoras Naturais/imunologia , Transdução de Sinais , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Anticorpos/farmacologia , Transporte Biológico/genética , Transporte Biológico/imunologia , Antígenos CD28/agonistas , Antígenos CD28/imunologia , Complexo CD3/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células , Colesterol/metabolismo , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-4/biossíntese , Interleucina-4/imunologia , Lipoproteínas/genética , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/citologia , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , TimoRESUMO
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
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
BACKGROUND: Adipose tissue (AT) is the body's largest free cholesterol reservoir and abundantly expresses ATP binding cassette transporter A1 (ABCA1), a key cholesterol transporter for high-density lipoprotein (HDL) biogenesis. However, the extent to which AT ABCA1 expression contributes to HDL biogenesis in vivo is unknown. METHODS AND RESULTS: Adipocyte-specific ABCA1 knockout mice (ABCA1(-A/-A)) were generated by crossing ABCA1(floxed) mice with aP2Cre transgenic mice. AT from ABCA1(-A/-A) mice had <10% of wild-type ABCA1 protein expression but normal hepatic and intestinal expression. Deletion of adipocyte ABCA1 resulted in a significant decrease in plasma HDL cholesterol (approximately 15%) and apolipoprotein A-I (approximately 13%) concentrations. AT from ABCA1(-A/-A) mice had a 2-fold increase in free cholesterol content compared with wild-type mice and failed to efflux cholesterol to apolipoprotein A-I. However, cholesterol efflux from AT to plasma HDL was similar for both genotypes of mice. Incubation of wild-type AT explants with apolipoprotein A-I resulted in the formation of multiple discrete-sized nascent HDL particles ranging in diameter from 7.1 to 12 nm; similar incubations with ABCA1(-A/-A) AT explants resulted in nascent HDL <8 nm. Plasma decay and tissue uptake of wild-type (125)I-HDL tracer were similar in both genotypes of recipient mice, suggesting that adipocyte ABCA1 deficiency reduces plasma HDL concentrations solely by reducing nascent HDL particle formation. CONCLUSIONS: We provide in vivo evidence that AT ABCA1-dependent cholesterol efflux and nascent HDL particle formation contribute to systemic HDL biogenesis and that AT ABCA1 expression plays an important role in adipocyte cholesterol homeostasis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Tecido Adiposo/metabolismo , Lipoproteínas HDL/biossíntese , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Adipócitos/metabolismo , Tecido Adiposo/citologia , Animais , Apolipoproteína A-I/sangue , Colesterol/metabolismo , Homeostase , Lipoproteínas HDL/sangue , Lipoproteínas HDL/química , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Tamanho da PartículaRESUMO
Cholesterol is a key component of cell membranes and is essential for cell growth and proliferation. How the accumulation of cellular cholesterol affects lymphocyte development and function is not well understood. We demonstrate that ATP-binding cassette transporter G1 (ABCG1) regulates cholesterol homeostasis in thymocytes and peripheral CD4 T cells. Our work is the first to describe a cell type in Abcg1-deficient mice with such a robust change in cholesterol content and the expression of cholesterol metabolism genes. Abcg1-deficient mice display increased thymocyte cellularity and enhanced proliferation of thymocytes and peripheral T lymphocytes in vivo. The absence of ABCG1 in CD4 T cells results in hyperproliferation in vitro, but only when cells are stimulated through the TCR. We hypothesize that cholesterol accumulation in Abcg1(-/-) T cells alters the plasma membrane structure, resulting in enhanced TCR signaling for proliferation. Supporting this idea, we demonstrate that B6 T cells pretreated with soluble cholesterol have a significant increase in proliferation. Cholesterol accumulation in Abcg1(-/-) CD4 T cells results in enhanced basal phosphorylation levels of ZAP70 and ERK1/2. Furthermore, inhibition of ERK phosphorylation in TCR-stimulated Abcg1(-/-) T cells rescues the hyperproliferative phenotype. We describe a novel mechanism by which cholesterol can alter signaling from the plasma membrane to affect downstream signaling pathways and proliferation. These results implicate ABCG1 as an important negative regulator of lymphocyte proliferation through the maintenance of cellular cholesterol homeostasis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proliferação de Células , Colesterol/metabolismo , Lipoproteínas/metabolismo , Transdução de Sinais/fisiologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Western Blotting , Contagem de Células , Membrana Celular/metabolismo , Citometria de Fluxo , Homeostase/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologiaRESUMO
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
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
Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for pre beta HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent pre beta HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that approximately 50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent pre beta HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with (125)I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Pre beta HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent pre beta HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with pre beta HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent pre beta HDL formation but does stimulate the formation of larger-sized pre beta HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto pre beta HDL during or after their formation by ABCA1.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteínas/metabolismo , Regulação da Expressão Gênica , Lipoproteínas de Alta Densidade Pré-beta/química , Lipoproteínas de Alta Densidade Pré-beta/metabolismo , Tamanho da Partícula , Transportador 1 de Cassete de Ligação de ATP , Sequência de Aminoácidos , Animais , Apolipoproteínas/química , Apolipoproteínas/genética , Apolipoproteínas/isolamento & purificação , Apolipoproteínas M , Carbonatos/química , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Humanos , Espaço Intracelular/metabolismo , Lipocalinas , Dados de Sequência MolecularRESUMO
Patients with Tangier disease exhibit extremely low plasma HDL concentrations resulting from mutations in the ATP-binding cassette, sub-family A, member 1 (ABCA1) protein. ABCA1 controls the rate-limiting step in HDL particle assembly by mediating efflux of cholesterol and phospholipid from cells to lipid-free apoA-I, which forms nascent HDL particles. ABCA1 is widely expressed; however, the specific tissues involved in HDL biogenesis are unknown. To determine the role of the liver in HDL biogenesis, we generated mice with targeted deletion of the second nucleotide-binding domain of Abca1 in liver only (Abca1(-L/-L)). Abca1(-L/-L) mice had total plasma and HDL cholesterol concentrations that were 19% and 17% those of wild-type littermates, respectively. In vivo catabolism of HDL apoA-I from wild-type mice or human lipid-free apoA-I was 2-fold higher in Abca1(-L/-L) mice compared with controls due to a 2-fold increase in the catabolism of apoA-I by the kidney, with no change in liver catabolism. We conclude that in chow-fed mice, the liver is the single most important source of plasma HDL. Furthermore, hepatic, but not extrahepatic, Abca1 is critical in maintaining the circulation of mature HDL particles by direct lipidation of hepatic lipid-poor apoA-I, slowing its catabolism by the kidney and prolonging its plasma residence time.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Apolipoproteína A-I/metabolismo , Rim/metabolismo , Lipoproteínas HDL/sangue , Doença de Tangier/genética , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Marcação de Genes , Genótipo , Hepatócitos/metabolismo , Humanos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Doença de Tangier/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.