RESUMEN
Background: Familial lecithin: cholesterol acyltransferase (LCAT) deficiency (FLD) is a severe inherited disease without effective treatment. Patients with FLD develop severe low HDL, corneal opacity, hemolytic anemia, and renal injury. Objective: We developed genetically modified adipocytes (GMAC) secreting LCAT (LCAT-GMAC) for ex vivo gene therapy. GMACs were prepared from the patient's adipocytes to express LCAT by retroviral gene transduction to secrete functional enzymes. This study aimed to evaluate the safety and efficacy of LCAT-GMAC implantation in an FLD patient. Methods: Proliferative preadipocytes were obtained from a patient using a ceiling culture and retrovirally transduced with LCAT. After obtaining enough cells by expansion culture of the transduced cells, the resulting LCAT-GMACs were implanted into a patient with FLD. To evaluate the safety and efficacy, we analyzed the outcome of the autologous implantation for 24 weeks of observation and subsequent 240 weeks of the follow-up periods. Results: This first-in-human autologous implantation of LCAT-GMACs was shown to be safe by evaluating adverse events. The LCAT-GMAC implantation increased serum LCAT activity by approximately 50% of the baseline and sustained over three years. Consistent with increased LCAT activity, intermediate-density lipoprotein (IDL) and free cholesterol levels of the small and very small HDL fractions decreased. We found the hemoglobin/haptoglobin complex in the hemolyzed pre-implantation sera of the patient. After one week of the implantation, the hemoglobin/haptoglobin complex almost disappeared. Immediately after the implantation, the patient's proteinuria decreased temporarily to mild levels and gradually increased to the baseline. At 48 weeks after implantation, the patient's proteinuria deteriorated with the development of mild hypertension. By the treatment with antihypertensives, the patient's blood pressure normalized. With the normalization of blood pressure, the proteinuria rapidly decreased to mild proteinuria levels. Conclusions: LCAT-GMAC implantation in a patient with FLD is shown to be safe and appears to be effective, in part, for treating anemia and proteinuria in FLD.
RESUMEN
Neuronal migration contributes to the establishment of mammalian brain. The extracellular protein Reelin sends signals to various downstream molecules by binding to its receptors, the apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor and exerts essential roles in the neuronal migration and formation of the layered neocortex. However, the cellular and molecular functions of Reelin signaling in the cortical development are not yet fully understood. Here, to gain insight into the role of Reelin signaling during cortical development, we examined the migratory behavior of Apoer2-deficient neurons in the developing brain. Stage-specific labeling of newborn neurons revealed that the neurons ectopically invaded the marginal zone (MZ) and that neuronal migration of both early- and late-born neurons was disrupted in the intermediate zone (IZ) in the Apoer2 KO mice. Rescue experiments showed that ApoER2 functions both in cell-autonomous and noncell-autonomous manners, that Rap1, integrin, and Akt are involved in the termination of migration beneath the MZ, and that Akt also controls neuronal migration in the IZ downstream of ApoER2. These data indicate that ApoER2 controls multiple processes in neuronal migration, including the early stage of radial migration and termination of migration beneath the MZ in the developing neocortex.
Asunto(s)
Movimiento Celular/fisiología , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Neuronas/metabolismo , Animales , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/crecimiento & desarrollo , Región CA1 Hipocampal/metabolismo , Corteza Cerebral/citología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Integrinas/metabolismo , Proteínas Relacionadas con Receptor de LDL/genética , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/citología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Reelina , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
In mammalian developing brain, neuronal migration is regulated by a variety of signaling cascades, including Reelin signaling. Reelin is a glycoprotein that is mainly secreted by Cajal-Retzius neurons in the marginal zone, playing essential roles in the formation of the layered neocortex via its receptors, apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR). However, the precise mechanisms by which Reelin signaling controls the neuronal migration process remain unclear. To gain insight into how Reelin signaling controls individual migrating neurons, we generated monoclonal antibodies against ApoER2 and VLDLR and examined the localization of Reelin receptors in the developing mouse cerebral cortex. Immunohistochemical analyses revealed that VLDLR is localized to the distal portion of leading processes in the marginal zone (MZ), whereas ApoER2 is mainly localized to neuronal processes and the cell membranes of multipolar cells in the multipolar cell accumulation zone (MAZ). These different expression patterns may contribute to the distinct actions of Reelin on migrating neurons during both the early and late migratory stages in the developing cerebral cortex.
Asunto(s)
Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Receptores de LDL/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Proteínas Relacionadas con Receptor de LDL/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de LDL/genética , Proteína Reelina , Serina Endopeptidasas/metabolismo , TransfecciónRESUMEN
BACKGROUND: Comparison of the reactivity of remnant-like lipoprotein particles (RLP) and LDL particles to LDL receptor and VLDL receptor has not been investigated. METHODS: LDL receptor- or VLDL receptor-transfected ldlA-7, HepG2 and L6 cells were used. Human LDL and rabbit ß-VLDL were isolated by ultracentrifugation. Human RLP was isolated using an immunoaffinity mixed gel. The effect of statin on lipoprotein receptors was examined. RESULTS: Both LDL receptor and VLDL receptor recognized RLP. In LDL receptor transfectants, RLP, ß-VLDL and LDL all bound to LDL receptor. Cold RLP competed efficiently with DiI-ß-VLDL; however, cold LDL competed weakly. In VLDL receptor transfectants, RLP and ß-VLDL bound to VLDL receptor, but not LDL. RLP bound to VLDL receptor with higher affinity than ß-VLDL because of higher apolipoprotein E in RLP. LDL receptor expression was induced in HepG2 by the low concentration of statin while VLDL receptor expression was induced in L6 myoblasts at higher concentration. CONCLUSIONS: RLP are bound to hepatic LDL receptor more efficiently than LDL, which may explain the mechanism by which statins prevent cardiovascular risk by primarily reducing plasma RLP rather than by reducing LDL. Additionally, a high-dose of statins also may reduce plasma RLP through muscular VLDL receptor.
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Colesterol/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lipoproteínas LDL/metabolismo , Lipoproteínas/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Triglicéridos/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glucosa/metabolismo , Células Hep G2 , Humanos , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Unión Proteica/efectos de los fármacos , Quinolinas/farmacología , Ratas , Especificidad por Sustrato , Activación Transcripcional/efectos de los fármacosRESUMEN
Triglyceride-rich lipoproteins (TGRLs) and low-density-lipoprotein (LDL) cholesterol are independent risk factors for coronary artery disease. We have previously proposed that the very low-density-lipoprotein (VLDL) receptor is one of the receptors required for foam cell formation by TGRLs in human macrophages. However, the VLDL receptor proteins have not been detected in atherosclerotic lesions of several animal models. Here we showed no VLDL receptor protein was detected in mouse macrophage cell lines (Raw264.7 and J774.2) or in mouse peritoneal macrophages in vitro. Furthermore, no VLDL receptor protein was detected in macrophages in atherosclerotic lesions of chow-fed apolipoprotein E-deficient or cholesterol-fed LDL receptor-deficient mice in vivo. In contrast, macrophage VLDL receptor protein was clearly detected in human macrophages in vitro and in atherosclerotic lesions in myocardial infarction-prone Watanabe-heritable hyperlipidemic (WHHLMI) rabbits in vivo. There are species differences in the localization of VLDL receptor protein in vitro and in vivo. Since VLDL receptor is expressed on macrophages in atheromatous plaques of both rabbit and human but not in mouse models, the mechanisms of atherogenesis and/or growth of atherosclerotic lesions in mouse models may be partly different from those of humans and rabbits.
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Macrófagos Peritoneales/metabolismo , Receptores de LDL/metabolismo , Animales , Humanos , Inmunohistoquímica , Macrófagos Peritoneales/química , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Conejos , Receptores de LDL/análisis , Especificidad de la EspecieRESUMEN
Acetate is activated to acetyl-CoA by acetyl-CoA synthetase 2 (AceCS2), a mitochondrial enzyme. Here, we report that the activation of acetate by AceCS2 has a specific and unique role in thermogenesis during fasting. In the skeletal muscle of fasted AceCS2(-/-) mice, ATP levels were reduced by 50% compared to AceCS2(+/+) mice. Fasted AceCS2(-/-) mice were significantly hypothermic and had reduced exercise capacity. Furthermore, when fed a low-carbohydrate diet, 4-week-old weaned AceCS2(-/-) mice also exhibited hypothermia accompanied by sustained hypoglycemia that led to a 50% mortality. Therefore, AceCS2 plays a significant role in acetate oxidation needed to generate ATP and heat. Furthermore, AceCS2(-/-) mice exhibited increased oxygen consumption and reduced weight gain on a low-carbohydrate diet. Our findings demonstrate that activation of acetate by AceCS2 plays a pivotal role in thermogenesis, especially under low-glucose or ketogenic conditions, and is crucially required for survival.
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Acetato CoA Ligasa/fisiología , Metabolismo Energético , Termogénesis/fisiología , Acetato CoA Ligasa/genética , Adenosina Trifosfato/metabolismo , Animales , Ayuno , Hipoglucemia/etiología , Hipotermia Inducida , Ratones , Ratones Noqueados , Consumo de OxígenoRESUMEN
Sex-determining region Y-box (SOX) 6 negatively regulates glucose-stimulated insulin secretion from beta-cells and is a down-regulated transcription factor in the pancreatic islet cells of hyperinsulinemic obese mice. To determine the contribution of SOX6 to insulin resistance, we analyzed the effects of SOX6 on cell proliferation. Small interfering RNA-mediated attenuation of SOX6 expression stimulated the proliferation of insulinoma INS-1E and NIH-3T3 cells, whereas retroviral overexpression resulted in inhibition of cell growth. Quantitative real time-PCR analysis revealed that the levels of cyclin D1 transcripts were markedly decreased by SOX6 overexpression. Luciferase-reporter assay with beta-catenin showed that SOX6 suppresses cyclin D1 promoter activities. In vitro binding experiments showed that the LZ/Q domain of SOX6 physically interacts with armadillo repeats 1-4 of beta-catenin. Furthermore, chromatin immunoprecipitation assay revealed that increased SOX6 expression significantly reduced the levels of acetylated histones H3 and H4 at the cyclin D1 promoter. By using a histone deacetylase (HDAC) inhibitor and co-immunoprecipitation analysis, we showed that SOX6 suppressed cyclin D1 activities by interacting withbeta-catenin and HDAC1. The data presented suggest that SOX6 may be an important factor in obesity-related insulin resistance.
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Ciclina D1/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Histona Desacetilasas/metabolismo , Células Secretoras de Insulina/fisiología , Factores de Transcripción/metabolismo , beta Catenina/metabolismo , Animales , División Celular/fisiología , Línea Celular Tumoral , Ciclina D1/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/fisiología , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/genética , Histona Desacetilasa 1 , Histonas/metabolismo , Humanos , Hiperinsulinismo/metabolismo , Hiperinsulinismo/fisiopatología , Resistencia a la Insulina/fisiología , Células Secretoras de Insulina/citología , Insulinoma , Riñón/citología , Leucina Zippers/fisiología , Ratones , Células 3T3 NIH , Obesidad/metabolismo , Obesidad/fisiopatología , Neoplasias Pancreáticas , Regiones Promotoras Genéticas/fisiología , Estructura Terciaria de Proteína , Ratas , Factores de Transcripción SOXD , Factores de Transcripción/química , Factores de Transcripción/genética , Transducción GenéticaRESUMEN
Although apolipoprotein E (apoE) is well known to play a major role in lipid metabolism, its role in glucose and energy homeostasis remains unclear. Herein, we established apoE-deficient genetically obese Ay (apoE(-/-);Ay/+) mice. ApoE deficiency in Ay mice prevented the development of obesity, with decreased fat accumulation in the liver and adipose tissues. ApoE(-/-);Ay/+ mice exhibited better glucose tolerance than apoE(+/+);Ay/+ mice. Insulin tolerance testing and hyperinsulinemic-euglycemic clamp study revealed marked improvement of insulin sensitivity, despite increased plasma free fatty acid levels. These metabolic phenotypes were reversed by adenoviral replenishment of apoE protein, indicating circulating apoE to be involved in increased adiposity and obesity-related metabolic disorders. Uptake of apoE-lacking VLDL into the liver and adipocytes was markedly inhibited, but adipocytes in apoE(-/-);Ay/+ mice exhibited normal differentiation, suggesting that apoE-dependent VLDL transport is involved in the development of obesity, i.e., surplus fat accumulation. Interestingly, apoE(-/-);Ay/+ mice exhibited decreased food intake and increased energy expenditure. Pair-feeding experiments indicate these phenomena to both contribute to the obesity-resistant phenotypes associated with apoE deficiency. Thus, apoE is involved in maintaining energy homeostasis. ApoE-dependent excess fat accumulation is a promising therapeutic target for the metabolic syndrome.
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Tejido Adiposo/anatomía & histología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/fisiología , Resistencia a la Insulina/fisiología , Tejido Adiposo/fisiología , Animales , Apolipoproteínas E/genética , Peso Corporal , Cistinil Aminopeptidasa/metabolismo , Ratones , Ratones Noqueados , Ratones Obesos , Fosfotirosina/metabolismo , Triglicéridos/metabolismoRESUMEN
In obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing beta-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca(2+) mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in beta-cells may contribute to the beta-cell adaptation in obesity-related insulin resistance.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Glucosa/farmacología , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas de Homeodominio/antagonistas & inhibidores , Hiperinsulinismo/metabolismo , Insulina/metabolismo , Obesidad/metabolismo , Transactivadores/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Acetilación , Adenosina Trifosfato/metabolismo , Animales , Movimiento Celular , Cromatina/metabolismo , Dieta , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/farmacología , Glucosa/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Hiperinsulinismo/genética , Insulina/genética , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Ratones , Ratones Obesos , Mitocondrias/metabolismo , Obesidad/genética , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción SOXD , Transactivadores/metabolismo , Transcripción GenéticaRESUMEN
The glucose-induced insulin secretion is fine-tuned by numerous factors. To systematically identify insulinotropic factors, we optimized a primary beta-cell-based functional assay to monitor intracellular Ca2+ flux ([Ca2+]i). By this assay system, we successfully identified several insulinotropic peptides including cholecystokinin, gastrin releasing peptide, vasopressin, and oxytocin from tissue extracts. Screening of an assortment of chemical compounds, we determined three novel insulin secretagogues: N-arachidonylglycine (NAGly), 3beta-(2-diethylamino-ethoxy) androstenone hydrochloride (U18666A), and 4-androstene-3,17-dione. The NAGly increased [Ca2+]i through stimulation of the voltage-dependent Ca2+ channels and it was dependent on extracellular glucose level. On the other hand, U18666A and 4-androstene-3,17-dione increased [Ca2+]i in the presence of K ATP channel opener diazoxide while it was inhibited by the presence of Ca2+ channel blocker nitrendipine, suggesting that their effects are independent of K ATP channel. These unique features will be useful for further development of insulinotropic factors and drugs for treating type 2 diabetes.
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Androstenodiona/farmacología , Androstenos/farmacología , Ácidos Araquidónicos/farmacología , Glicina/análogos & derivados , Insulina/metabolismo , Animales , Calcio/metabolismo , Glicina/farmacología , Secreción de Insulina , Masculino , Ratones , Ratones Endogámicos ICR , Ratas , Ratas WistarRESUMEN
Hyperlipidemia is a common feature of diabetes and is related to cardiovascular disease. The very low-density lipoprotein receptor (VLDL-R) is a member of the low-density lipoprotein receptor (LDL-R) family. It binds and internalizes triglyceride-rich lipoproteins with high specificity. We examined the etiology of hyperlipidemia in the insulin-deficient state. VLDL-R expression in heart and skeletal muscle were measured in rats with streptozotocin (STZ)-induced diabetes. STZ rats showed severe hyperlipidemia on d 21 and 28, with a dramatic decline in VLDL-R protein in skeletal muscle (>90%), heart (approximately 50%) and a loss of adipose tissues itself on d 28. The reduction of VLDL-R protein in skeletal muscle could not be explained simply by a decrease at the transcriptional level, because a dissociation between VLDL-R protein and mRNA expression was observed. The expression of LDL-R and LDL-R-related protein in liver showed no consistent changes. Furthermore, no effect on VLDL-triglyceride production in liver was observed in STZ rats. A decrease in postheparin plasma lipoprotein lipase activity started on d 7 and continued to d 28 at the 50% level even though severe hyperlipidemia was detected only on d 21 and 28. In rat myoblast cells, serum deprivation for 24 h induced a reduction in VLDL-R proteins. Insulin (10(-6) m), but not IGF-I (10 ng/ml), restored the decreased VLDL-R proteins by serum deprivation. These results suggest that the combination of VLDL-R deficiency and reduced plasma lipoprotein lipase activity may be responsible for severe hyperlipidemia in insulin-deficient diabetes.
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Diabetes Mellitus Experimental/fisiopatología , Insulina/fisiología , Receptores de LDL/fisiología , Tejido Adiposo/fisiopatología , Animales , HDL-Colesterol/sangre , LDL-Colesterol/sangre , VLDL-Colesterol/sangre , Corazón/fisiopatología , Hiperlipidemias/etiología , Lipoproteínas/metabolismo , Masculino , Músculo Esquelético/fisiopatología , ARN/genética , ARN/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Receptores de LDL/deficiencia , Receptores de LDL/genética , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Triglicéridos/metabolismoRESUMEN
The low-density lipoprotein (LDL) receptor (LDLR) is a crucial role for binding and uptaking apolipoprotein (apo) B-containing lipoproteins, such as very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and LDL. The defect function of the LDLR causes familial hypercholesterolemia (FH), the phenotype of which is elevated plasma cholesterol and premature coronary heart disease (CHD). In the present study, we characterize the role of the cysteine residue of the ligand-binding domain of the LDLR. The mutant LDLR protein of cysteine for serine at codon 25 (25S-LDLR) was expressed in Chinese hamster ovary (CHO) cell line, ldl-A7. By Western blot analysis, the 25S-LDLR was detected with monoclonal antibody IgG-12D10, which reacts with the linker site of the LDLR but not with IgG-C7, which reacts with the NH2 terminus of the receptor. The 25S-LDLR bound LDL similarly to the wild-type LDLR, but the rate of uptake of LDL by the mutant receptor was only about half of that by the wild-type receptor. In contrast, the 25S-LDLR bound and internalized beta VLDL more avidly than LDL. These results suggest that the fourth cysteine residue of the first ligand-binding domain of the LDLR might be important for the internalization of atherogenic lipoproteins by vascular cells despite reduced LDL uptake, leading to atherosclerosis and premature cardiovascular disease.
Asunto(s)
Cisteína/metabolismo , Hiperlipoproteinemia Tipo II/metabolismo , Lipoproteínas VLDL/metabolismo , Receptores de LDL/metabolismo , Animales , Western Blotting , Células CHO , Clonación Molecular , Cricetinae , Cricetulus , Cartilla de ADN , Citometría de Flujo , Humanos , Ligandos , Mutación/genética , Estructura Terciaria de Proteína , Receptores de LDL/genéticaRESUMEN
The very low-density lipoprotein (VLDL) receptor is a member of the low-density lipoprotein (LDL) receptor family. In vitro and in vivo studies have shown that VLDL receptor binds triglyceride (TG)-rich lipoproteins but not LDL, and functions as a peripheral remnant lipoprotein receptor. VLDL receptor is expressed abundantly in fatty acid-active tissues (heart, skeletal muscle and fat), the brain and macrophages. It is likely that VLDL receptor functions in concert with lipoprotein lipase (LPL), which hydrolyses TG in VLDL and chylomicron. In contrast to the LDL receptor, VLDL receptor binds apolipoprotein (apo) E2/2 VLDL particles as well as apoE3/3 VLDL, and the expression is not down-regulated by intracellular lipoproteins. Recently, various functions of the VLDL receptor have been reported in lipoprotein metabolism, metabolic syndrome/atherosclerosis, cardiac fatty acid metabolism, neuronal migration and angiogenesis/tumor growth. Gene therapy of VLDL receptor into the liver showed a benefit effect for lipoprotein metabolism in both LDL receptor knockout and apoE mutant mice. Beyond its function as a peripheral lipoprotein receptor, possibilities of its physiological function have been extended to include signal transduction, angiogenesis and tumor growth.
Asunto(s)
Receptores de LDL/fisiología , Animales , Apolipoproteínas E/genética , Arteriosclerosis/genética , Arteriosclerosis/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Clonación Molecular , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Terapia Genética/métodos , Humanos , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/terapia , Lipoproteínas VLDL/metabolismo , Síndrome Metabólico/genética , Síndrome Metabólico/metabolismo , Mutación , Proteínas del Tejido Nervioso , Receptores de Lipoproteína/fisiología , Proteína Reelina , Serina Endopeptidasas , Transducción de SeñalRESUMEN
By consensus, the acyl-CoA synthetase (ACS) community, with the advice of the human and mouse genome nomenclature committees, has revised the nomenclature for the mammalian long-chain acyl-CoA synthetases. ACS is the family root name, and the human and mouse genes for the long-chain ACSs are termed ACSL1,3-6 and Acsl1,3-6, respectively. Splice variants of ACSL3, -4, -5, and -6 are cataloged. Suggestions for naming other family members and for the nonmammalian acyl-CoA synthetases are made.
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Acilcoenzima A , Coenzima A Ligasas/genética , Terminología como Asunto , Animales , Genes , Humanos , Ratones , Familia de MultigenesRESUMEN
Acetyl-CoA synthetase 2 (AceCS2) produces acetyl-CoA for oxidation through the citric acid cycle in the mitochondrial matrix. AceCS2 is highly expressed in the skeletal muscle and is robustly induced by fasting. Quantification of AceCS2 transcripts both in C2C12 and human myotubes indicated that fasting-induced AceCS2 gene expression appears to be independent on insulin action. Characterization of 5'-flanking region of the mouse AceCS2 gene demonstrates that Krüppel-like factor 15 (KLF15) plays a key role in the trans-activation of the AceCS2 gene. Deletion and mutation analyses of AceCS2 promoter region revealed that the most proximal KLF site is a curtail site for the trans-activation of the AceCS2 gene by KLF15. Using Sp-null Drosophila SL2 cells, we showed that the combination of KLF15 and Sp1 resulted in a synergistic activation of the AceCS2 promoter. Mutation analyses of three GC-boxes in the AceCS2 promoter indicated that the GC-box, located 8 bases downstream of the most proximal KLF15 site, is the most important GC-box in the synergistic trans-activation of the AceCS2 gene by KLF15 and Sp1. GST pull-down assays showed that KLF15 interacts with Sp1 in vitro. Quantification of various KLF transcripts revealed that 48 h fasting robustly induced the KLF15 transcripts in the skeletal muscle. Together with the trans-activation of the AceCS2 promoter, it is suggested that fasting-induced AceCS2 expression is largely contributed by KLF15. Furthermore, KLF15 overexpression induced the levels of AceCS2 transcripts both in myoblasts and in myotubes, indicating that AceCS2 gene expression in vivo is indeed induced by KLF15.
Asunto(s)
Acetato CoA Ligasa/genética , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional , Acetato CoA Ligasa/metabolismo , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Ciclo del Ácido Cítrico , Clonación Molecular , Análisis Mutacional de ADN , ADN Complementario/metabolismo , Proteínas de Unión al ADN , Drosophila , Eliminación de Gen , Genes Reporteros , Glutatión Transferasa/metabolismo , Humanos , Insulina/metabolismo , Factores de Transcripción de Tipo Kruppel , Masculino , Ratones , Ratones Endogámicos ICR , Modelos Genéticos , Datos de Secuencia Molecular , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/genética , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factor de Transcripción Sp1/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , TransfecciónRESUMEN
The VLDL (very low density lipoprotein) receptor is a member of the LDL (low density lipoprotein) receptor family. The VLDL receptor binds apolipoprotein (apo) E but not apo B, and is expressed in fatty acid active tissues (heart, muscle, adipose) and macrophages abundantly. Lipoprotein lipase (LPL) modulates the binding of triglyceride (TG)-rich lipoprotein particles to the VLDL receptor. By the unique ligand specificity, VLDL receptor practically appeared to function as IDL (intermediate density lipoprotein) and chylomicron remnant receptor in peripheral tissues in concert with LPL. In contrast to LDL receptor, the VLDL receptor expression is not down regulated by lipoproteins. Recently several possible functions of the VLDL receptor have been reported in lipoprotein metabolism, atherosclerosis, obesity/insulin resistance, cardiac fatty acid metabolism and neuronal migration. The gene therapy of VLDL receptor into the LDL receptor knockout mice liver showed a benefit effect for lipoprotein metabolism and atherosclerosis. Further researches about the VLDL receptor function will be needed in the future.
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Ácidos Grasos/metabolismo , Lipoproteínas/metabolismo , Receptores de LDL/fisiología , Animales , Arteriosclerosis/metabolismo , Movimiento Celular , Terapia Genética , Humanos , Ligandos , Modelos Biológicos , Miocardio/metabolismo , Neuronas/metabolismo , Unión Proteica , Receptores de LDL/metabolismoRESUMEN
LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had approximately 60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were approximately 3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced atherosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.
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Apolipoproteínas E/fisiología , Arteriosclerosis/genética , Grasas de la Dieta/metabolismo , Hipercolesterolemia/genética , Receptores de LDL/fisiología , Animales , Apolipoproteínas E/genética , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Proteínas Relacionadas con Receptor de LDL , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Ratones Noqueados , Receptores de LDL/genéticaRESUMEN
A Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) plays an essential role in bone accrual and eye development. Here, we show that LRP5 is also required for normal cholesterol and glucose metabolism. The production of mice lacking LRP5 revealed that LRP5 deficiency led to increased plasma cholesterol levels in mice fed a high-fat diet, because of the decreased hepatic clearance of chylomicron remnants. In addition, when fed a normal diet, LRP5-deficient mice showed a markedly impaired glucose tolerance. The LRP5-deficient islets had a marked reduction in the levels of intracellular ATP and Ca(2+) in response to glucose, and thereby glucose-induced insulin secretion was decreased. The intracellular inositol 1,4,5-trisphosphate (IP3) production in response to glucose was also reduced in LRP5-- islets. Real-time PCR analysis revealed a marked reduction of various transcripts for genes involved in glucose sensing in LRP5-- islets. Furthermore, exposure of LRP5++ islets to Wnt-3a and Wnt-5a stimulates glucose-induced insulin secretion and this stimulation was blocked by the addition of a soluble form of Wnt receptor, secreted Frizzled-related protein-1. In contrast, LRP5-deficient islets lacked the Wnt-3a-stimulated insulin secretion. These data suggest that WntLRP5 signaling contributes to the glucose-induced insulin secretion in the islets.
Asunto(s)
Colesterol/sangre , Glucosa/farmacología , Hipercolesterolemia/genética , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Receptores de LDL/genética , Animales , Glucemia/metabolismo , Calcio/metabolismo , Quilomicrones/metabolismo , Grasas de la Dieta , Genes Esenciales , Intolerancia a la Glucosa/sangre , Intolerancia a la Glucosa/genética , Hipercolesterolemia/sangre , Inositol 1,4,5-Trifosfato/metabolismo , Insulina/sangre , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Proteínas Relacionadas con Receptor de LDL , Hígado/metabolismo , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Receptores de LDL/deficiencia , Transcripción GenéticaRESUMEN
By expression cloning using fluorescent-labeled high density lipoprotein (HDL), we isolated two clones that conferred the cell surface binding of HDL. Nucleotide sequence of the two clones revealed that one corresponds to scavenger receptor class B, type 1 (SRBI) and the other encoded a novel protein with 228 amino acids. The primary structure of the newly identified HDL-binding protein resembles GPI-anchored proteins consisting of an N-terminal signal sequence, an acidic region with a cluster of aspartate and glutamate residues, an Ly-6 motif highly conserved among the lymphocyte antigen family, and a C-terminal hydrophobic region. This newly identified HDL-binding protein designated GPI-anchored HDL-binding protein 1 (GPI-HBP1), was susceptible to phosphatidylinositol-specific phospholipase C treatment and binds HDL with high affinity (calculated K(d) = 2-3 microg/ml). Similar to SRBI, GPI-HBP1 mediates selective lipid uptake but not the protein component of HDL. Among various ligands for SRBI, HDL was most preferentially bound to GPI-HBP1. In contrast to SRBI, GPI-HBP1 lacked HDL-dependent cholesterol efflux. The GPI-HBP1 transcripts were detected with the highest levels in heart and, to a much lesser extent, in lung and liver. In situ hybridization revealed the accumulation of GPI-HBP1 transcripts in cardiac muscle cells, hepatic Kupffer cells and sinusoidal endothelium, and bronchial epithelium and alveolar macrophages in the lung.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Lipoproteínas HDL/metabolismo , Receptores de Lipoproteína/química , Receptores de Lipoproteína/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Northern Blotting , Células CHO , Colesterol/metabolismo , Clonación Molecular , Cricetinae , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Hibridación in Situ , Cinética , Macrófagos del Hígado , Ligandos , Hígado/metabolismo , Pulmón/metabolismo , Ratones , Datos de Secuencia Molecular , Miocardio/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Factores de Tiempo , Distribución Tisular , TransfecciónRESUMEN
The VLDL (very low-density lipoprotein) receptor is a peripheral lipoprotein receptor expressing in fatty acid active tissues abundantly. In the Balb/c fasting mice, VLDL receptor as well as LPL (lipoprotein lipase), FAT (fatty acid translocase)/CD36, H-FABP (heart-type fatty acid-binding protein), ACS (acyl-CoA synthetase) and LCAD (long-chain acyl-CoA dehydrogenase) expressions increased. An electron microscopic examination indicated the lipid droplets that accumulated in the hearts of fasting Balb/c mice. During the development of SD (Sprague-Dawley) rats, VLDL receptor, LPL, FAT/CD36, H-FABP, ACS, and LCAD mRNAs concomitantly increased with growth. However, PK (pyruvate kinase) mRNA expression was negligible. In cultured neonatal rat cardiomyocytes, VLDL receptor expression increased with days in culture. Oil red-O staining showed that cardiomyocytes after 7 days in culture (when the VLDL receptor protein is present) accumulated beta-migrating VLDL. Thereby, we showed that the cardiac VLDL receptor pathway for delivery of remnant lipoprotein particles might be part of a cardiac fatty acid metabolism.