RESUMEN
OBJECTIVE: Obesity and its associated comorbidities represent a global health challenge with a need for well-tolerated, effective, and mechanistically diverse pharmaceutical interventions. Oxyntomodulin is a gut peptide that activates the glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) and reduces bodyweight by increasing energy expenditure and reducing energy intake in humans. Here we describe the pharmacological profile of the novel glucagon receptor (GCGR)/GLP-1 receptor (GLP-1R) dual agonist BI 456906. METHODS: BI 456906 was characterized using cell-based in vitro assays to determine functional agonism. In vivo pharmacological studies were performed using acute and subchronic dosing regimens to demonstrate target engagement for the GCGR and GLP-1R, and weight lowering efficacy. RESULTS: BI 456906 is a potent, acylated peptide containing a C18 fatty acid as a half-life extending principle to support once-weekly dosing in humans. Pharmacological doses of BI 456906 provided greater bodyweight reductions in mice compared with maximally effective doses of the GLP-1R agonist semaglutide. BI 456906's superior efficacy is the consequence of increased energy expenditure and reduced food intake. Engagement of both receptors in vivo was demonstrated via glucose tolerance, food intake, and gastric emptying tests for the GLP-1R, and liver nicotinamide N-methyltransferase mRNA expression and circulating biomarkers (amino acids, fibroblast growth factor-21) for the GCGR. The dual activity of BI 456906 at the GLP-1R and GCGR was supported using GLP-1R knockout and transgenic reporter mice, and an ex vivo bioactivity assay. CONCLUSIONS: BI 456906 is a potent GCGR/GLP-1R dual agonist with robust anti-obesity efficacy achieved by increasing energy expenditure and decreasing food intake.
Asunto(s)
Péptido 1 Similar al Glucagón , Receptores de Glucagón , Animales , Humanos , Ratones , Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Oxintomodulina/farmacología , Péptidos/farmacología , Péptidos/metabolismo , Receptores de Glucagón/metabolismoRESUMEN
Apathy, deficiency of motivation including willingness to exert effort for reward, is a common symptom in many psychiatric and neurological disorders, including depression and schizophrenia. Despite improved understanding of the neurocircuitry and neurochemistry underlying normal and deficient motivation, there is still no approved pharmacological treatment for such a deficiency. GPR139 is an orphan G protein-coupled receptor expressed in brain regions which contribute to the neural circuitry that controls motivation including effortful responding for reward, typically sweet gustatory reward. The GPR139 agonist TAK-041 is currently under development for treatment of negative symptoms in schizophrenia which include apathy. To date, however, there are no published preclinical data regarding its potential effect on reward motivation or deficiencies thereof. Here we report in vitro evidence confirming that TAK-041 increases intracellular Ca2+ mobilization and has high selectivity for GPR139. In vivo, TAK-041 was brain penetrant and showed a favorable pharmacokinetic profile. It was without effect on extracellular dopamine concentration in the nucleus accumbens. In addition, TAK-041 did not alter the effort exerted to obtain sweet gustatory reward in rats that were moderately food deprived. By contrast, TAK-041 increased the effort exerted to obtain sweet gustatory reward in mice that were only minimally food deprived; furthermore, this effect of TAK-041 occurred both in control mice and in mice in which deficient effortful responding was induced by chronic social stress. Overall, this study provides preclinical evidence in support of GPR139 agonism as a molecular target mechanism for treatment of apathy.
Asunto(s)
Motivación , Roedores , Animales , Dopamina/metabolismo , Gastos en Salud , Ratones , Proteínas del Tejido Nervioso/farmacología , Ratas , Receptores Acoplados a Proteínas G , Recompensa , Roedores/metabolismoRESUMEN
The anorectic action of the pancreatic hormone amylin is mainly mediated through the area postrema (AP). Amylin activates AP neurons using a heterodimeric receptor (AMY) composed of the calcitonin receptor (CTR) and the receptor activity modifying protein (RAMP 1, 2 or 3). The aim of the following experiments is to test the effects of the long acting amylin analogue (LAAMA) in RAMP1/3 knock-out (KO) male mice and in neuronal CTR KO Nestin-CreCTR male mice. In vitro, LAAMA exerted an equipotent effect on CTR and AMYs that was maintained across species. Following one week of 45% high fat diet, WT, RAMP1/3 KO and Nestin-CreCTR mice were injected daily for one week with vehicle or LAAMA. LAAMA decreased body weight gain in WT and in RAMP1/3 KO mice suggesting that RAMP1/3 are not necessary for LAAMA-induced effects. However, LAAMA was not able to produce any body lowering and anorectic effects in Nestin-CreCTR mice. This was accompanied by the absence of any c-Fos signal in the AP opposite to WT control mice. Together, these results suggest that LAAMA's effects are mainly mediated through CTR rather than specific AMY. The study of LAAMA or any amylin receptor agonist in different receptor KO mouse models helps disentangle the underlying mechanisms used by these molecules.
Asunto(s)
Receptores de Calcitonina , Animales , Área Postrema , Polipéptido Amiloide de los Islotes Pancreáticos , Ratones , Proteínas Proto-Oncogénicas c-fosRESUMEN
The peptide hormone amylin reduces food intake and body weight and is an attractive candidate target for novel pharmacotherapies to treat obesity. However, the short half-life of native amylin and amylin analogs like pramlintide limits these compounds' potential utility in promoting sustained negative energy balance. Here, we evaluate the ability of the novel long-acting amylin/calcitonin receptor agonist ZP5461 to reduce feeding and body weight in rats, and also test the role of calcitonin receptors (CTRs) in the dorsal vagal complex (DVC) of the hindbrain in the energy balance effects of chronic ZP5461 administration. Acute dose-response studies indicate that systemic ZP5461 (0.5-3 nmol/kg) robustly suppresses energy intake and body weight gain in chow- and high-fat diet (HFD)-fed rats. When HFD-fed rats received chronic systemic administration of ZP5461 (1-2 nmol/kg), the compound initially produced reductions in energy intake and weight gain but failed to produce sustained suppression of intake and body weight. Using virally mediated knockdown of DVC CTRs, the ability of chronic systemic ZP5461 to promote early reductions in intake and body weight gain was determined to be mediated in part by activation of DVC CTRs, implicating the DVC as a central site of action for ZP5461. Future studies should address other dosing regimens of ZP5461 to determine whether an alternative dose/frequency of administration would produce more sustained body weight suppression.
Asunto(s)
Agonistas de los Receptores de Amilina/farmacología , Depresores del Apetito/farmacología , Ingestión de Alimentos/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Receptores de Calcitonina/agonistas , Receptores de Polipéptido Amiloide de Islotes Pancreáticos/efectos de los fármacos , Rombencéfalo/efectos de los fármacos , Nervio Vago/efectos de los fármacos , Aumento de Peso/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Ingestión de Energía/efectos de los fármacos , Masculino , Ratas Sprague-Dawley , Receptores de Calcitonina/genética , Receptores de Calcitonina/metabolismo , Receptores de Polipéptido Amiloide de Islotes Pancreáticos/genética , Receptores de Polipéptido Amiloide de Islotes Pancreáticos/metabolismo , Rombencéfalo/metabolismo , Transducción de Señal , Factores de Tiempo , Nervio Vago/metabolismoRESUMEN
Dipeptidyl peptidase IV (DPP-IV) inhibitors improve glycemic control by prolonging the action of glucagon-like peptide-1 (GLP-1). In contrast to GLP-1 analogues, DPP-IV inhibitors are weight-neutral. DPP-IV cleavage of PYY and NPY gives rise to PYY3-36 and NPY3-36 which exert potent anorectic action by stimulating Y2 receptor (Y2R) function. This invites the possibility that DPP-IV inhibitors could be weight-neutral by preventing conversion of PYY/NPY to Y2R-selective peptide agonists. We therefore investigated whether co-administration of an Y2R-selective agonist could unmask potential weight lowering effects of the DDP-IV inhibitor linagliptin. Male diet-induced obese (DIO) mice received once daily subcutaneous treatment with linagliptin (3 mg/kg), a Y2R-selective PYY3-36 analogue (3 or 30 nmol/kg) or combination therapy for 14 days. While linagliptin promoted marginal weight loss without influencing food intake, the PYY3-36 analogue induced significant weight loss and transient suppression of food intake. Both compounds significantly improved oral glucose tolerance. Because combination treatment did not further improve weight loss and glucose tolerance in DIO mice, this suggests that potential negative modulatory effects of DPP-IV inhibitors on endogenous Y2R peptide agonist activity is likely insufficient to influence weight homeostasis. Weight-neutrality of DPP-IV inhibitors may therefore not be explained by counter-regulatory effects on PYY/NPY responses.
Asunto(s)
Inhibidores de la Dipeptidil-Peptidasa IV , Linagliptina/farmacología , Receptores de Neuropéptido Y , Animales , Masculino , Ratones , Ratones ObesosRESUMEN
Despite increased knowledge of nutrient intake regulation and energy homeostasis, treatment options for obesity remain limited. Food reward consists of two branches: gustatory and post-ingestive nutritive information. Drosophila lacking dSLC5A11 (sodium/glucose cotransporter 6-SGLT6) prefer L-glucose over D-glucose independently of their state of satiety. Human SGLT6 is an active transporter of myo-inositol and D-glucose. We investigated expression of SGLT6 in human tissue and found a significant expression in the small intestine and brain. The preference between a metabolizable and a non-metabolizable sugar was tested in 3 mouse models with a selective and potent SGLT6 inhibitor. No influence on sugar preference was seen with SGLT6 inhibition. These studies suggest that SGLT6 does not play a significant role in nutrient sensing in mammals.
Asunto(s)
Fármacos Antiobesidad/farmacología , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/metabolismo , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Simportadores/antagonistas & inhibidores , Simportadores/metabolismo , Animales , Fármacos Antiobesidad/farmacocinética , Fármacos Antiobesidad/uso terapéutico , Células CACO-2 , Preferencias Alimentarias/efectos de los fármacos , Glucosa/metabolismo , Células HEK293 , Humanos , Inositol/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Terapia Molecular DirigidaRESUMEN
The inhibitory effect of anti-obesity drugs on energy intake (EI) is counter-acted by feedback regulation of the appetite control circuit leading to drug tolerance. This complicates the design and interpretation of EI studies in rodents that are used for anti-obesity drug development. Here, we investigated a synthetic long-acting analogue of the appetite-suppressing peptide hormone amylin (LAMY) in lean and diet-induced obese (DIO) rats. EI and body weight (BW) were measured daily and LAMY concentrations in plasma were assessed using defined time points following subcutaneous administration of the LAMY at different dosing regimens. Overall, 6 pharmacodynamic (PD) studies including a total of 173 rats were considered in this evaluation. Treatment caused a dose-dependent reduction in EI and BW, although multiple dosing indicated the development of tolerance over time. This behavior could be adequately described by a population model including homeostatic feedback of EI and a turnover model describing the relationship between EI and BW. The model was evaluated by testing its ability to predict BW loss in a toxicology study and was utilized to improve the understanding of dosing regimens for obesity therapy. As such, the model proved to be a valuable tool for the design and interpretation of rodent studies used in anti-obesity drug development.
Asunto(s)
Peso Corporal/efectos de los fármacos , Ingestión de Energía/efectos de los fármacos , Polipéptido Amiloide de los Islotes Pancreáticos/farmacología , Polipéptido Amiloide de los Islotes Pancreáticos/farmacocinética , Animales , Fármacos Antiobesidad/farmacocinética , Fármacos Antiobesidad/farmacología , Dieta Alta en Grasa/métodos , Femenino , Masculino , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Previous studies have shown that mice with defects in cellular cholesterol efflux show hematopoietic stem cell (HSPC) and myeloid proliferation, contributing to atherogenesis. We hypothesized that the combination of hypercholesterolemia and defective cholesterol efflux would promote HSPC expansion and leukocytosis more prominently than either alone. We crossed Ldlr(-/-) with Apoa1(-/-) mice and found that compared to Ldlr(-/-) mice, Ldlr(-/-)/Apoa1(+/-) mice, with similar LDL-cholesterol levels but reduced HDL-cholesterol (HDL-C) levels, had expansion of HSPCs, monocytosis and neutrophilia. Ex vivo studies showed that HSPCs expressed high levels of Ldlr, Scarb1 (Srb1), and Lrp1 and were able to take up both native and oxidized LDL. Native LDL directly stimulated HSPC proliferation, while co-incubation with reconstituted HDL attenuated this effect. We also assessed the impact of HDL-C levels on monocytes in children with familial hypercholesterolemia (FH) (n = 49) and found that subjects with the lowest level of HDL-C, had increased monocyte counts compared to the mid and higher HDL-C levels. Overall, HDL-C was inversely correlated with the monocyte count. These data suggest that in mice, a balance of cholesterol uptake and efflux mechanisms may be one factor in driving HSPC proliferation and monocytosis. Higher monocyte counts in children with FH and low HDL-cholesterol suggest a similar pattern in humans.
Asunto(s)
Aterosclerosis/metabolismo , HDL-Colesterol/metabolismo , Células Madre Hematopoyéticas/citología , Hiperlipoproteinemia Tipo II/metabolismo , Monocitos/citología , Adolescente , Animales , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Aterosclerosis/inmunología , Aterosclerosis/patología , Proliferación Celular , Niño , LDL-Colesterol/metabolismo , Femenino , Células Madre Hematopoyéticas/metabolismo , Humanos , Hiperlipoproteinemia Tipo II/inmunología , Hiperlipoproteinemia Tipo II/patología , Lipoproteínas LDL/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismoRESUMEN
RATIONALE: Plasma high-density lipoprotein levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is attributable to the ability of high-density lipoprotein to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. OBJECTIVE: To assess the role of macrophage cholesterol efflux pathways in atherogenesis. METHODS AND RESULTS: We developed mice with efficient deletion of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) in macrophages (MAC-ABC(DKO) mice) but not in hematopoietic stem or progenitor populations. MAC-ABC(DKO) bone marrow (BM) was transplanted into Ldlr(-/-) recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared with controls. On the Western-type diet, MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice had disproportionate atherosclerosis, considering they also had lower very low-density lipoprotein/low-density lipoprotein cholesterol levels than controls. ABCA1/G1-deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, Western-type diet-fed MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice displayed monocytosis and neutrophilia in the absence of hematopoietic stem and multipotential progenitor cells proliferation. Mechanistic studies revealed increased expressions of machrophage colony stimulating factor and granulocyte colony stimulating factor in splenic macrophage foam cells, driving BM monocyte and neutrophil production. CONCLUSIONS: These studies show that macrophage deficiency of ABCA1/G1 is proatherogenic likely by promoting plaque inflammation and uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Aterosclerosis/inmunología , Lipoproteínas/genética , Lipoproteínas/inmunología , Vasculitis/inmunología , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/metabolismo , Alimentación Animal , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Trasplante de Médula Ósea , Colesterol en la Dieta/metabolismo , Células Espumosas/inmunología , Células Espumosas/metabolismo , Células Espumosas/patología , Lipoproteínas/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Noqueados , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/patología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Receptores de LDL/genética , Receptores de LDL/metabolismo , Bazo/patología , Vasculitis/genética , Vasculitis/patologíaRESUMEN
Leukocytosis is associated with increased cardiovascular disease risk in humans and develops in hypercholesterolemic atherosclerotic animal models. Leukocytosis is associated with the proliferation of hematopoietic stem and multipotential progenitor cells (HSPCs) in mice with deficiencies of the cholesterol efflux-promoting ABC transporters ABCA1 and ABCG1 in BM cells. Here, we have determined the role of endogenous apolipoprotein-mediated cholesterol efflux pathways in these processes. In Apoeâ»/â» mice fed a chow or Western- type diet, monocytosis and neutrophilia developed in association with the proliferation and expansion of HSPCs in the BM. In contrast, Apoa1â»/â» mice showed no monocytosis compared with controls. ApoE was found on the surface of HSPCs, in a proteoglycan-bound pool, where it acted in an ABCA1- and ABCG1-dependent fashion to decrease cell proliferation. Accordingly, competitive BM transplantation experiments showed that ApoE acted cell autonomously to control HSPC proliferation, monocytosis, neutrophilia, and monocyte accumulation in atherosclerotic lesions. Infusion of reconstituted HDL and LXR activator treatment each reduced HSPC proliferation and monocytosis in Apoeâ»/â» mice. These studies suggest a specific role for proteoglycanbound ApoE at the surface of HSPCs to promote cholesterol efflux via ABCA1/ABCG1 and decrease cell proliferation, monocytosis, and atherosclerosis. Although endogenous apoA-I was ineffective, pharmacologic approaches to increasing cholesterol efflux suppressed stem cell proliferative responses.
Asunto(s)
Apolipoproteínas E/fisiología , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/fisiología , Monocitos/patología , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Apolipoproteína A-I/deficiencia , Apolipoproteína A-I/genética , Apolipoproteína A-I/fisiología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Proliferación Celular , Colesterol/metabolismo , Modelos Animales de Enfermedad , Humanos , Leucocitosis/patología , Leucocitosis/fisiopatología , Lipoproteínas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Cardiovasculares , Proteoglicanos/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores de LDL/fisiologíaRESUMEN
RATIONALE: Reduced plasma cholesterol and increased high-density lipoprotein (HDL) levels promote regression of atherosclerosis, in a process characterized by lipid unloading and emigration of macrophages from lesions. In contrast free cholesterol loading of macrophages leads to imbalanced Rac1/Rho activities and impaired chemotaxis. OBJECTIVE: To study the role of HDL and the ATP-binding cassette transporters ABCA1 and ABCG1 in modulating the chemotaxis of macrophages. METHODS AND RESULTS: Abca1(-/-)Abcg1(-/-) mouse macrophages displayed profoundly impaired chemotaxis both in a Transwell chamber assay and in the peritoneal cavity of wild-type (WT) mice. HDL reversed impaired chemotaxis in free cholesterol-loaded WT macrophages but was without effect in Abca1(-/-)Abcg1(-/-) cells, whereas cyclodextrin was effective in both. Abca1(-/-)Abcg1(-/-) macrophages had markedly increased Rac1 activity and increased association of Rac1 with the plasma membrane (PM). Their defective chemotaxis was reversed by a Rac1 inhibitor. To gain a better understanding of the role of transporters in PM cholesterol movement, we measured transbilayer PM sterol distribution. In WT macrophages, the majority of cholesterol was located on the inner leaflet, whereas on upregulation of transporters by liver X receptor activation, PM sterol was shifted to the outer leaflet, where it could be removed by HDL. Abca1(-/-)Abcg1(-/-) macrophages showed increased PM sterol content and defective redistribution of sterol to the outer leaflet. CONCLUSIONS: Deletion of ABCA1 and ABCG1 causes an increased cholesterol content on the inner leaflet of the PM, associated with increased Rac1 PM localization, activation, and impairment of migration. ABCA1 and ABCG1 facilitate macrophage chemotaxis by promoting PM transbilayer cholesterol movement and may contribute to the ability of HDL to promote regression of atherosclerosis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Movimiento Celular/fisiología , Lipoproteínas/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Neuropéptidos/metabolismo , Transducción de Señal/fisiología , Proteínas de Unión al GTP rac/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/genética , Actinas/metabolismo , Animales , Membrana Celular/metabolismo , Células Cultivadas , Quimiotaxis/fisiología , Colesterol/metabolismo , Guanosina Trifosfato/metabolismo , Lipoproteínas/genética , Ratones , Ratones Noqueados , Modelos Animales , Proteína de Unión al GTP rac1RESUMEN
OBJECTIVE: To examine the effects of treatments with niacin or anacetrapib (an inhibitor of cholesteryl ester transfer protein) on the ability of high-density lipoprotein (HDL) to promote net cholesterol efflux and reduce toll-like receptor-mediated inflammation in macrophages. METHODS AND RESULTS: A total of 18 patients received niacin, 2 g/d, for 4 weeks; 20 patients received anacetrapib, 300 mg/d, for 8 weeks; and 2 groups (n=4 and n=5 patients) received placebo. HDL samples were isolated by polyethylene glycol precipitation or ultracentrifugation, tested for the ability to promote cholesterol efflux in cholesterol-loaded THP-I or mouse peritoneal macrophages, or used to pretreat macrophages, followed by lipopolysaccharide exposure. HDL cholesterol levels were increased by 30% in response to niacin and by approximately 100% in response to anacetrapib. Niacin treatment increased HDL-mediated net cholesterol efflux from foam cells, primarily by increasing HDL concentration, whereas anacetrapib treatment increased cholesterol efflux by both increasing HDL concentration and causing increased efflux at matched HDL concentrations. The increased efflux potential of anacetrapib-HDL was more prominent at higher HDL cholesterol concentrations (>12 microg/mL), which was associated with an increased content of lecithin-cholesterol acyltransferase (LCAT) and apolipoprotein E and completely dependent on the expression of ATP binding cassette transporters (ABCA1 and ABCG1). Potent antiinflammatory effects of HDL were observed at low HDL concentrations (3 to 20 microg/mL) and were partly dependent on the expression of ABCA1 and ABCG1. All HDL preparations showed similar antiinflammatory effects, proportionate to the HDL cholesterol concentration. CONCLUSIONS: Niacin treatment caused a moderate increase in the ability of HDL to promote net cholesterol efflux, whereas inhibition of cholesteryl ester transfer protein via anacetrapib led to a more dramatic increase in association with enhanced particle functionality at higher HDL concentrations. All HDLs exhibited potent ability to suppress macrophage toll-like receptor 4-mediated inflammatory responses, in a process partly dependent on cholesterol efflux via ABCA1 and ABCG1.
Asunto(s)
Antiinflamatorios/uso terapéutico , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , HDL-Colesterol/sangre , Colesterol/sangre , Dislipidemias/tratamiento farmacológico , Hipolipemiantes/uso terapéutico , Inflamación/prevención & control , Macrófagos/metabolismo , Niacina/uso terapéutico , Oxazolidinonas/uso terapéutico , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antiinflamatorios/efectos adversos , Células Cultivadas , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Ensayos Clínicos como Asunto , Dislipidemias/sangre , Dislipidemias/inmunología , Células Espumosas/metabolismo , Humanos , Hipolipemiantes/efectos adversos , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Lipoproteínas/deficiencia , Lipoproteínas/genética , Lipoproteínas/metabolismo , Macrófagos/inmunología , Ratones , Ratones Noqueados , Niacina/efectos adversos , Oxazolidinonas/efectos adversos , Factores de Tiempo , Receptor Toll-Like 4/metabolismo , Resultado del TratamientoRESUMEN
RATIONALE: Antiatherogenic effects of plasma high-density lipoprotein (HDL) include the ability to inhibit apoptosis of macrophage foam cells. The ATP-binding cassette transporters ABCA1 and ABCG1 have a major role in promoting cholesterol efflux from macrophages to apolipoprotein A-1 and HDL and are upregulated during the phagocytosis of apoptotic cells (efferocytosis). OBJECTIVE: The goal of this study was to determine the roles of ABCA1 and ABCG1 in preserving the viability of macrophages during efferocytosis. METHODS AND RESULTS: We show that despite similar clearance of apoptotic cells, peritoneal macrophages from Abca1(-/-)Abcg1(-/-), Abcg1(-/-), and, to a lesser extent, Abca1(-/-) mice are much more prone to apoptosis during efferocytosis compared to wild-type cells. Similar findings were observed following incubations with oxidized phospholipids, and the ability of HDL to protect against oxidized phospholipid-induced apoptosis was markedly reduced in Abca1(-/-)Abcg1(-/-) and Abcg1(-/-) cells. These effects were independent of any role of ABCA1 and ABCG1 in mediating oxidized phospholipid efflux but were reversed by cyclodextrin-mediated cholesterol efflux. The apoptotic response observed in Abca1(-/-)Abcg1(-/-) macrophages after oxidized phospholipid exposure or engulfment of apoptotic cells was dependent on an excessive oxidative burst secondary to enhanced assembly of NADPH oxidase (NOX)2 complexes, leading to sustained Jnk activation which turned on the apoptotic cell death program. Increased NOX2 assembly required Toll-like receptors 2/4 and MyD88 signaling, which are known to be enhanced in transporter deficient cells in a lipid raft-dependent fashion. CONCLUSIONS: We identified a new beneficial role of ABCA1, ABCG1 and HDL in dampening the oxidative burst and preserving viability of macrophages following exposure to oxidized phospholipids and/or apoptotic cells.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Apoptosis/fisiología , Lipoproteínas/fisiología , Macrófagos Peritoneales/fisiología , Estrés Oxidativo/fisiología , Fagocitosis/fisiología , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Supervivencia Celular/fisiología , Células Cultivadas , Lipoproteínas/genética , Lipoproteínas HDL/fisiología , MAP Quinasa Quinasa 4/fisiología , Macrófagos Peritoneales/citología , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Factor 88 de Diferenciación Mieloide/fisiología , NADPH Oxidasa 2 , NADPH Oxidasas/fisiología , Receptor Toll-Like 2/fisiología , Receptor Toll-Like 4/fisiologíaRESUMEN
Elevated leukocyte cell numbers (leukocytosis), and monocytes in particular, promote atherosclerosis; however, how they become increased is poorly understood. Mice deficient in the adenosine triphosphate-binding cassette (ABC) transporters ABCA1 and ABCG1, which promote cholesterol efflux from macrophages and suppress atherosclerosis in hypercholesterolemic mice, displayed leukocytosis, a transplantable myeloproliferative disorder, and a dramatic expansion of the stem and progenitor cell population containing Lin(-)Sca-1(+)Kit+ (LSK) in the bone marrow. Transplantation of Abca1(-/-) Abcg1(-/-) bone marrow into apolipoprotein A-1 transgenic mice with elevated levels of high-density lipoprotein (HDL) suppressed the LSK population, reduced leukocytosis, reversed the myeloproliferative disorder, and accelerated atherosclerosis. The findings indicate that ABCA1, ABCG1, and HDL inhibit the proliferation of hematopoietic stem and multipotential progenitor cells and connect expansion of these populations with leukocytosis and accelerated atherosclerosis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Aterosclerosis/fisiopatología , Colesterol/metabolismo , Células Madre Hematopoyéticas/fisiología , Leucocitosis/fisiopatología , Lipoproteínas HDL/metabolismo , Lipoproteínas/metabolismo , Células Progenitoras Mieloides/fisiología , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/terapia , Trasplante de Médula Ósea , Proliferación Celular , Células Cultivadas , Hipercolesterolemia/metabolismo , Leucocitosis/metabolismo , Leucocitosis/terapia , Lipoproteínas/genética , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Células Madre Multipotentes/fisiología , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/fisiopatología , Trastornos Mieloproliferativos/terapia , Fenotipo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Interleucina-3/metabolismo , Transducción de SeñalRESUMEN
Holo-high density lipoprotein (HDL) particle uptake, besides selective lipid uptake, constitutes an alternative pathway to regulate cellular cholesterol homeostasis. In the current study, the cellular path of holo-HDL particles was investigated in human liver carcinoma cells (HepG2) using combined light and electron microscopical methods. The apolipoprotein moiety of HDL was visualized with different markers: horseradish peroxidase, colloidal gold and the fluorochrome Alexa(568), used in fluorescence microscopy and after photooxidation correlatively at the ultrastructural level. Time course experiments showed a rapid uptake of holo-HDL particles, an accumulation in endosomal compartments, with a plateau after 1-2 h of continuous uptake, and a clearance 1-2 h upon replacement by unlabeled HDL. Correlative microscopy, using HDL-Alexa(568)-driven diaminobenzidine (DAB) photooxidation, identified the fluorescent organelles as DAB-positive multivesicular bodies (MVBs) in the electron microscope; their luminal contents but not the internal vesicles were stained. Labeled MVBs increased in numbers and changed shapes along with the duration of uptake, from polymorphic organelles with multiple surface domains and differently shaped protrusions dominating at early times of uptake to compact bodies with mainly tubular appendices and densely packed vesicles after later times. Differently shaped and labeled surface domains and appendices, as revealed by three dimensional reconstructions, as well as images of homotypic fusions indicate the dynamics of the HDL-positive MVBs. Double staining visualized by confocal microscopy, along with the electron microscopic data, shows that holo-HDL particles after temporal storage in MVBs are only to a minor degree transported to lysosomes, which suggests that different mechanisms are involved in cellular HDL clearance, including resecretion.
Asunto(s)
Endocitosis , Endosomas/metabolismo , Lipoproteínas HDL/metabolismo , Endosomas/química , Oro/química , Oro/metabolismo , Células Hep G2 , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Lipoproteínas HDL/química , Tamaño de la PartículaRESUMEN
BACKGROUND: Two macrophage ABC transporters, ABCA1 and ABCG1, have a major role in promoting cholesterol efflux from macrophages. Peritoneal macrophages deficient in ABCA1, ABCG1, or both show enhanced expression of inflammatory and chemokine genes. This study was undertaken to elucidate the mechanisms and consequences of enhanced inflammatory gene expression in ABC transporter-deficient macrophages. METHODS AND RESULTS: Basal and lipopolysaccharide-stimulated thioglycollate-elicited peritoneal macrophages showed increased inflammatory gene expression in the order Abca1(-/-)Abcg1(-/-)>Abcg1(-/-)>Abca1(-/-)>wild-type. The increased inflammatory gene expression was abolished in macrophages deficient in Toll-like receptor 4 (TLR4) or MyD88/TRIF. TLR4 cell surface concentration was increased in Abca1(-/-)Abcg1(-/-)>Abcg1(-/-)> Abca1(-/-)> wild-type macrophages. Treatment of transporter-deficient cells with cyclodextrin reduced and cholesterol-cyclodextrin loading increased inflammatory gene expression. Abca1(-/-)Abcg1(-) bone marrow-derived macrophages showed enhanced inflammatory gene responses to TLR2, TLR3, and TLR4 ligands. To assess in vivo relevance, we injected intraperitoneally thioglycollate in Abcg1(-/-) bone marrow-transplanted, Western diet-fed, Ldlr-deficient mice. This resulted in a profound inflammatory infiltrate in the adventitia and necrotic core region of atherosclerotic lesions, consisting primarily of neutrophils. CONCLUSIONS: The results suggest that high-density lipoprotein and apolipoprotein A-1 exert anti-inflammatory effects by promoting cholesterol efflux via ABCG1 and ABCA1 with consequent attenuation of signaling via Toll-like receptors. In response to a peripheral inflammatory stimulus, atherosclerotic lesions containing Abcg1(-/-) macrophages experience an inflammatory "echo," suggesting a possible mechanism of plaque destabilization in subjects with low high-density lipoprotein levels.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Aterosclerosis/inmunología , Colesterol/metabolismo , Lipoproteínas/genética , Receptor Toll-Like 4/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/inmunología , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Aterosclerosis/metabolismo , Aterosclerosis/fisiopatología , Regulación de la Expresión Génica/inmunología , Ligandos , Lipopolisacáridos/farmacología , Lipoproteínas/inmunología , Lipoproteínas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Microdominios de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Mutantes , Neutrófilos/inmunología , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transducción de Señal/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
Plasma HDL levels are inversely related to the incidence of atherosclerotic disease. Some of the atheroprotective effects of HDL are likely mediated via preservation of EC function. Whether the beneficial effects of HDL on ECs depend on its involvement in cholesterol efflux via the ATP-binding cassette transporters ABCA1 and ABCG1, which promote efflux of cholesterol and oxysterols from macrophages, has not been investigated. To address this, we assessed endothelial function in Abca1(-/-), Abcg1(-/-), and Abca1(-/-)Abcg1(-/-) mice fed either a high-cholesterol diet (HCD) or a Western diet (WTD). Non-atherosclerotic arteries from WTD-fed Abcg1(-/-) and Abca1(-/-)Abcg1(-/-) mice exhibited a marked decrease in endothelium-dependent vasorelaxation, while Abca1(-/-) mice had a milder defect. In addition, eNOS activity was reduced in aortic homogenates generated from Abcg1(-/-) mice fed either a HCD or a WTD, and this correlated with decreased levels of the active dimeric form of eNOS. More detailed analysis indicated that ABCG1 was expressed primarily in ECs, and that these cells accumulated the oxysterol 7-ketocholesterol (7-KC) when Abcg1(-/-) mice were fed a WTD. Consistent with these data, ABCG1 had a major role in promoting efflux of cholesterol and 7-KC in cultured human aortic ECs (HAECs). Furthermore, HDL treatment of HAECs prevented 7-KC-induced ROS production and active eNOS dimer disruption in an ABCG1-dependent manner. Our data suggest that ABCG1 and HDL maintain EC function in HCD-fed mice by promoting efflux of cholesterol and 7-oxysterols and preserving active eNOS dimer levels.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Colesterol en la Dieta/efectos adversos , Colesterol/metabolismo , Endotelio Vascular/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Cetocolesteroles/metabolismo , Lipoproteínas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Óxido Nítrico Sintasa de Tipo III/metabolismoRESUMEN
High-density lipoprotein (HDL) has been identified as a potential target in the treatment of atherosclerotic vascular disease. The failure of torcetrapib, an inhibitor of cholesteryl ester transfer protein (CETP) that markedly increased HDL levels in a clinical trial, has called into doubt the efficacy of HDL elevation. Recent analysis suggests that failure may have been caused by off-target toxicity and that HDL is functional and promotes regression of atherosclerosis. New studies highlight the central importance of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 in reducing macrophage foam cell formation, inflammation, and atherosclerosis. A variety of approaches to increasing HDL may eventually be successful in treating atherosclerosis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Aterosclerosis/terapia , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Animales , Aterosclerosis/metabolismo , HumanosRESUMEN
This study compares the roles of ABCG1 and scavenger receptor class B type I (SR-BI) singly or together in promoting net cellular cholesterol efflux to plasma HDL containing active LCAT. In transfected cells, SR-BI promoted free cholesterol efflux to HDL, but this was offset by an increased uptake of HDL cholesteryl ester (CE) into cells, resulting in no net efflux. Coexpression of SR-BI with ABCG1 inhibited the ABCG1-mediated net cholesterol efflux to HDL, apparently by promoting the reuptake of CE from medium. However, ABCG1-mediated cholesterol efflux was not altered in cholesterol-loaded, SR-BI-deficient (SR-BI(-/-)) macrophages. Briefly cultured macrophages collected from SR-BI(-/-) mice loaded with acetylated LDL in the peritoneal cavity did exhibit reduced efflux to HDL. However, this was attributable to reduced expression of ABCG1 and ABCA1, likely reflecting increased macrophage cholesterol efflux to apolipoprotein E-enriched HDL during loading in SR-BI(-/-) mice. In conclusion, cellular SR-BI does not promote net cholesterol efflux from cells to plasma HDL containing active LCAT as a result of the reuptake of HDL-CE into cells. Previous findings of increased atherosclerosis in mice transplanted with SR-BI(-/-) bone marrow probably cannot be explained by a defect in macrophage cholesterol efflux.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , HDL-Colesterol/sangre , Colesterol/metabolismo , Lipoproteínas LDL/sangre , Lipoproteínas/metabolismo , Macrófagos Peritoneales/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Receptores Depuradores de Clase B/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Animales , Línea Celular , Ésteres del Colesterol/sangre , Proteínas de Unión al ADN/metabolismo , Humanos , Lipoproteínas HDL/sangre , Receptores X del Hígado , Ratones , Ratones Mutantes , Receptores Nucleares Huérfanos , ARN Interferente Pequeño/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , TransfecciónRESUMEN
Recently, we showed that holo HDL particle uptake and resecretion occur in physiologically relevant cell lines and that HDL uptake is mediated by scavenger receptor class B type I (SR-BI). Furthermore, we established that HDL resecretion is accompanied by [(3)H]cholesterol efflux. This study shows that HDL uptake and resecretion occur even when LDL uptake and cholesterol trafficking are disturbed. First, we used a set of inhibitors that block cholesterol transport out of the lysosome: chloroquine, imipramine, U18666A, and monensin. In all cases, HDL retroendocytosis occurred and HDL resecretion mediated [(3)H]cholesterol efflux, although to a lesser extent. Second, cell lines carrying somatic mutations in intracellular cholesterol transport were used: CHO 2-2 and CHO 3-6 cells accumulated LDL-derived lipid in the lysosome but showed all components of HDL retroendocytosis. SR-BI overexpression increased HDL uptake and resecretion and [(3)H]cholesterol efflux in these mutant cells. Finally, we used Niemann-Pick type C (NPC) patient fibroblast cells, which carry a defect in cholesterol transfer out of the lysosome. NPC fibroblast cells accumulate cholesterol in the lysosome as a result of a mutation in the NPC1 gene. Despite disturbed intracellular cholesterol transfer, NPC fibroblast cells exhibited HDL retroendocytosis and [(3)H]cholesterol efflux via HDL resecretion, although to a lesser extent. Thus, [(3)H]cholesterol efflux via HDL resecretion is independent of the cholesterol uptake pathway via the LDL receptor and may be an alternative way to remove excess cholesterol.