RESUMEN
Tirzepatide, a glucose-dependent insulinotropic polypeptide/glucagon-like peptide 1 receptor (GIPR/GLP-1R) agonist, has, in clinical trials, demonstrated greater reductions in glucose, body weight, and triglyceride levels compared with selective GLP-1R agonists in people with type 2 diabetes (T2D). However, cellular mechanisms by which GIPR agonism may contribute to these improved efficacy outcomes have not been fully defined. Using human adipocyte and mouse models, we investigated how long-acting GIPR agonists regulate fasted and fed adipocyte functions. In functional assays, GIPR agonism enhanced insulin signaling, augmented glucose uptake, and increased the conversion of glucose to glycerol in a cooperative manner with insulin; however, in the absence of insulin, GIPR agonists increased lipolysis. In diet-induced obese mice treated with a long-acting GIPR agonist, circulating triglyceride levels were reduced during oral lipid challenge, and lipoprotein-derived fatty acid uptake into adipose tissue was increased. Our findings support a model for long-acting GIPR agonists to modulate both fasted and fed adipose tissue function differentially by cooperating with insulin to augment glucose and lipid clearance in the fed state while enhancing lipid release when insulin levels are reduced in the fasted state.
Asunto(s)
Adipocitos , Polipéptido Inhibidor Gástrico , Receptores de la Hormona Gastrointestinal , Animales , Humanos , Masculino , Ratones , Adipocitos/metabolismo , Adipocitos/efectos de los fármacos , Polipéptido Inhibidor Gástrico/metabolismo , Polipéptido Inhibidor Gástrico/farmacología , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 2 Similar al Glucagón , Glucosa/metabolismo , Insulina/metabolismo , Lipólisis/efectos de los fármacos , Ratones Endogámicos C57BL , Nutrientes/metabolismo , Obesidad/metabolismo , Obesidad/tratamiento farmacológico , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de la Hormona Gastrointestinal/agonistas , Transducción de Señal/efectos de los fármacos , Triglicéridos/metabolismoRESUMEN
Apolipoprotein AV (APOA5) lowers plasma triglyceride (TG) levels by binding to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppressing its capacity to inhibit lipoprotein lipase (LPL) catalytic activity and its ability to detach LPL from binding sites within capillaries. However, the sequences in APOA5 that are required for suppressing ANGPTL3/8 activity have never been defined. A clue to the identity of those sequences was the presence of severe hypertriglyceridemia in two patients harboring an APOA5 mutation that truncates APOA5 by 35 residues ("APOA5Δ35"). We found that wild-type (WT) human APOA5, but not APOA5Δ35, suppressed ANGPTL3/8's ability to inhibit LPL catalytic activity. To pursue that finding, we prepared a mutant mouse APOA5 protein lacking 40 C-terminal amino acids ("APOA5Δ40"). Mouse WT-APOA5, but not APOA5Δ40, suppressed ANGPTL3/8's capacity to inhibit LPL catalytic activity and sharply reduced plasma TG levels in mice. WT-APOA5, but not APOA5Δ40, increased intracapillary LPL levels and reduced plasma TG levels in Apoa5-/- mice (where TG levels are high and intravascular LPL levels are low). Also, WT-APOA5, but not APOA5Δ40, blocked the ability of ANGPTL3/8 to detach LPL from cultured cells. Finally, an antibody against a synthetic peptide corresponding to the last 26 amino acids of mouse APOA5 reduced intracapillary LPL levels and increased plasma TG levels in WT mice. We conclude that C-terminal sequences in APOA5 are crucial for suppressing ANGPTL3/8 activity in vitro and for regulating intracapillary LPL levels and plasma TG levels in vivo.
Asunto(s)
Apolipoproteínas , Lipoproteína Lipasa , Ratones , Humanos , Animales , Proteínas Similares a la Angiopoyetina/genética , Proteínas Similares a la Angiopoyetina/metabolismo , Lipoproteína Lipasa/metabolismo , Proteína 3 Similar a la Angiopoyetina , Aminoácidos , Triglicéridos/metabolismo , Apolipoproteína A-V/genéticaRESUMEN
Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition. To understand how LPL binds ANGPTL3/8 and ApoA5 blocks this interaction, we used hydrogen-deuterium exchange mass-spectrometry and molecular modeling to map binding sites of LPL and ApoA5 on ANGPTL3/8. Remarkably, we found that LPL and ApoA5 both bound a unique ANGPTL3/8 epitope consisting of N-terminal regions of ANGPTL3 and ANGPTL8 that are unmasked upon formation of the ANGPTL3/8 complex. We further used ANGPTL3/8 as an immunogen to develop an antibody targeting this same epitope. After refocusing on antibodies that bound ANGPTL3/8, as opposed to ANGPTL3 or ANGPTL8 alone, we utilized bio-layer interferometry to select an antibody exhibiting high-affinity binding to the desired epitope. We revealed an ANGPTL3/8 leucine zipper-like motif within the anti-ANGPTL3/8 epitope, the LPL-inhibitory region, and the ApoA5-interacting region, suggesting the mechanism by which ApoA5 lowers TG is via competition with LPL for the same ANGPTL3/8-binding site. Supporting this hypothesis, we demonstrate that the anti-ANGPTL3/8 antibody potently blocked ANGPTL3/8-mediated LPL inhibition in vitro and dramatically lowered TG levels in vivo. Together, these data show that an anti-ANGPTL3/8 antibody targeting the same leucine zipper-containing epitope recognized by LPL and ApoA5 markedly decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition.
Asunto(s)
Lipoproteína Lipasa , Hormonas Peptídicas , Proteína 3 Similar a la Angiopoyetina , Proteína 8 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina/metabolismo , Apolipoproteína A-V , Epítopos , Humanos , Leucina Zippers , Lipoproteína Lipasa/metabolismo , Hormonas Peptídicas/metabolismo , Triglicéridos/metabolismoRESUMEN
A recent report described a novel mechanism of action for an anti-proprotein convertase subtilisin-kexin type 9 (PCSK9) monoclonal antibody (LY3015014, or LY), wherein the antibody has improved potency and duration of action due to the PCSK9 epitope for LY binding. Unlike other antibodies, proteolysis of PCSK9 can occur when LY is bound to PCSK9. We hypothesized that this allowance of PCSK9 cleavage potentially improves LY efficiency through two pathways, namely lack of accumulation of intact PCSK9 and reduced clearance of LY. A quantitative modeling approach is necessary to further understand this novel mechanism of action. We developed a mechanism-based model to characterize the relationship between antibody pharmacokinetics, PCSK9 and LDL cholesterol levels in animals, and used the model to better understand the underlying drivers for the improved efficiency of LY. Simulations suggested that the allowance of cleavage of PCSK9 resulting in a lack of accumulation of intact PCSK9 is the major driver of the improved potency and durability of LY. The modeling reveals that this novel 'proteolysis-permitting' mechanism of LY is a means by which an efficient antibody can be developed with a total antibody dosing rate that is lower than the target production rate. We expect this engineering approach may be applicable to other targets and that the mathematical models presented herein will be useful in evaluating similar approaches.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Modelos Teóricos , Inhibidores de PCSK9 , Animales , LDL-Colesterol/metabolismo , Humanos , Macaca fascicularis , Ratones , Proteolisis/efectos de los fármacosRESUMEN
Lilly PCSK9 antibody LY3015014 (LY) is a monoclonal antibody (mAb) that neutralizes proprotein convertase subtilisin-kexin type 9 (PCSK9). LY decreases LDL cholesterol in monkeys and, unlike other PCSK9 mAbs, does not cause an accumulation of intact PCSK9 in serum. Comparing the epitope of LY with other clinically tested PCSK9 mAbs, it was noted that the LY epitope excludes the furin cleavage site in PCSK9, whereas other mAbs span this site. In vitro exposure of PCSK9 to furin resulted in degradation of PCSK9 bound to LY, whereas cleavage was blocked by other mAbs. These other mAbs caused a significant accumulation of serum PCSK9 and displayed a shorter duration of LDL-cholesterol lowering than LY when administered to mice expressing the WT human PCSK9. In mice expressing a noncleavable variant of human PCSK9, LY behaved like a cleavage-blocking mAb, in that it caused significant PCSK9 accumulation, its duration of LDL lowering was reduced, and its clearance (CL) from serum was accelerated. Thus, LY neutralizes PCSK9 and allows its proteolytic degradation to proceed, which limits PCSK9 accumulation, reduces the CL rate of LY, and extends its duration of action. PCSK9 mAbs with this property are likely to achieve longer durability and require lower doses than mAbs that cause antigen to accumulate.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticolesterolemiantes/farmacología , Proproteína Convertasas/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Anticolesterolemiantes/química , Anticolesterolemiantes/farmacocinética , LDL-Colesterol/sangre , Evaluación Preclínica de Medicamentos , Estabilidad de Medicamentos , Furina/química , Semivida , Humanos , Hipercolesterolemia/tratamiento farmacológico , Macaca fascicularis , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proproteína Convertasa 9 , Proproteína Convertasas/inmunología , Unión Proteica , Proteolisis , Serina Endopeptidasas/inmunología , Resultado del TratamientoRESUMEN
This letter describes the discovery and SAR optimization of tetrazoyl tetrahydroquinoline derivatives as potent CETP inhibitors. Compound 6m exhibited robust HDL-c increase in hCETP/hApoA1 double transgenic model and favorable pharmacokinetic properties.
Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Diseño de Fármacos , Hipolipemiantes/síntesis química , Quinolinas/química , Tetrazoles/síntesis química , Animales , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol/genética , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , HDL-Colesterol/efectos de los fármacos , HDL-Colesterol/metabolismo , Perros , Femenino , Hipolipemiantes/química , Hipolipemiantes/farmacocinética , Inyecciones Intravenosas , Masculino , Ratones , Ratones Transgénicos , Quinolinas/síntesis química , Quinolinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Tetrazoles/química , Tetrazoles/farmacocinéticaRESUMEN
This Letter describes the discovery and SAR optimization of 1,5-tetrahydronaphthyridines, a new class of potent CETP inhibitors. The effort led to the identification of 21b and 21d with in vitro human plasma CETP inhibitory activity in the nanomolar range (IC(50)=23 and 22nM, respectively). Both 21b and 21d exhibited robust HDL-c increase in hCETP/hApoA1 dual heterozygous mice model.
Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Naftiridinas/farmacología , Animales , HDL-Colesterol , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Ratones , Naftiridinas/síntesis química , Relación Estructura-ActividadRESUMEN
Cholesteryl ester transfer protein (CETP) catalyses the exchange of cholesteryl ester and triglyceride between HDL and apoB containing lipoprotein particles. The role of CETP in modulating plasma HDL cholesterol levels in humans is well established and there have been significant efforts to develop CETP inhibitors to increase HDL cholesterol for the treatment of coronary artery disease. These efforts, however, have been hampered by the fact that most CETP inhibitors either have low potency or have undesirable side effects. In this study, we describe a novel benzazepine compound evacetrapib (LY2484595), which is a potent and selective inhibitor of CETP both in vitro and in vivo. Evacetrapib inhibited human recombinant CETP protein (5.5 nM IC(50)) and CETP activity in human plasma (36 nM IC(50)) in vitro. In double transgenic mice expressing human CETP and apoAI, evacetrapib exhibited an ex vivo CETP inhibition ED(50) of less than 5 mg/kg at 8 h post oral dose and significantly elevated HDL cholesterol. Importantly, no blood pressure elevation was observed in rats dosed with evacetrapib at high exposure multiples compared with the positive control, torcetrapib. In addition, in a human adrenal cortical carcinoma cell line (H295R cells), evacetrapib did not induce aldosterone or cortisol biosynthesis whereas torcetrapib dramatically induced aldosterone and cortisol biosynthesis. Our data indicate that evacetrapib is a potent and selective CETP inhibitor without torcetrapib-like off-target liabilities. Evacetrapib is currently in phase II clinical development.
Asunto(s)
Benzodiazepinas/efectos adversos , Benzodiazepinas/farmacología , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , HDL-Colesterol/sangre , Aldosterona/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratas , Especificidad por SustratoRESUMEN
Peroxisome proliferator-activated receptor alpha (PPARalpha) belongs to the nuclear receptor superfamily that regulates multiple target genes involved in lipid metabolism. Cholesterol ester transfer protein (CETP) is a secreted glycoprotein that modifies high-density lipoprotein (HDL) particles. In humans, plasma CETP activity is inversely correlated with HDL cholesterol levels. We report here that PPARalpha agonists increase CETP mRNA, protein and accordingly its activity. In a human CETP transgenic animal model harboring the natural flanking regions (Jiang et al. in J Clin Investigat 90:1290-1295, 1992), both fenofibrate and a specific synthetic PPARalpha agonist LY970 elevated human CETP mRNA in liver, serum protein and CETP activity. In hamsters, the endogenous liver CETP mRNA level and the serum CETP activity were dose-dependently upregulated by fenofibrate. In addition Wy14643, a PPARalpha agonist, also significantly elevated CETP mRNA and activity. In a carcinoma cell line of hepatic origin, HepG2 cells, overexpression of PPARalpha resulted in increased CETP mRNA and agonist treatment further elevated CETP mRNA levels. We conclude that PPARalpha agonists upregulate CETP expression and activity and may play an important role in PPARalpha (agonist mediated HDL cholesterol homeostasis in humans.
Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , PPAR alfa/agonistas , PPAR alfa/metabolismo , Animales , Células Cultivadas , Proteínas de Transferencia de Ésteres de Colesterol/efectos de los fármacos , Proteínas de Transferencia de Ésteres de Colesterol/genética , Cricetinae , Fenofibrato/farmacología , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , PPAR alfa/genética , ARN Mensajero/biosíntesisRESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that is known to reduce hepatic low-density lipoprotein receptor (LDLR) levels and increase plasma LDL cholesterol. It is not clear, however, whether secreted PCSK9 degrades extrahepatic LDLRs. We present evidence that recombinant PCSK9, either injected intravenously into or expressed in the liver of C57BL/6 mice, significantly reduced LDLR levels in multiple extrahepatic tissues. During the initial characterization, we found that injected human recombinant PCSK9 at 30 microg/mouse had a half-life of 15 min in serum in mice. Hepatic LDLR levels were reduced within 30min and the degradation of hepatic LDLR reached the maximum 2h after the initial protein injection. Endocytosis of PCSK9 in liver occurred within 5min of protein injection and internalized PCSK9 was only barely detectable within 1h. When extrahepatic LDLRs were examined by Western blotting analysis, we found significant reductions of LDLRs in multiple extrahepatic tissues including lung, adipose and kidney along with the more dramatic reduction of LDLRs in liver. These studies were further extended using adenoviral expression of human PCSK9 in C57BL/6 mice to demonstrate that PCSK9 produced in liver impacted extrahepatic tissue LDLR levels as well. Taken together, our studies indicate that secreted PCSK9 can potentially impact extrahepatic tissue cholesterol homeostasis by regulating extrahepatic tissue LDLR levels.
Asunto(s)
LDL-Colesterol/metabolismo , Hígado/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Adenoviridae/genética , Animales , Catálisis , LDL-Colesterol/sangre , Humanos , Inyecciones Intravenosas , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Proproteína Convertasa 9 , Proproteína Convertasas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Serina Endopeptidasas/genética , Serina Endopeptidasas/farmacocinéticaRESUMEN
Prebeta1 HDL is the initial plasma acceptor of cell-derived cholesterol in reverse cholesterol transport. Recently, small amphipathic peptides composed of D-amino acids have been shown to mimic apolipoprotein A-I (apoA-I) as a precursor for HDL formation. ApoA-I mimetic peptides have been proposed to stimulate the formation of prebeta1 HDL and increase reverse cholesterol transport in apoE-null mice. The existence of a monoclonal antibody (MAb 55201) and a corresponding ELISA method that is selective for the detection of the prebeta(1) subclass of HDL provides a means of establishing a correlation between apoA-I mimetic dose and prebeta1 HDL formation in human plasma. Using this prebeta1 HDL ELISA, we demonstrate marked apoA-I mimetic dose-dependent prebeta1 HDL formation in human plasma. These results correlated with increases in band density of the plasma prebeta1 HDL, when observed by Western blotting, as a function of increased apoA-I mimetic concentration. Increased prebeta1 HDL formation was observed after as little as 1 min and was maximal within 1 h. Together, these data suggest that a high-throughput prebeta1 HDL ELISA provides a way to quantitatively measure a key component of the reverse cholesterol transport pathway in human plasma, thus providing a possible method for the identification of apoA-I mimetic molecules.
Asunto(s)
Apolipoproteína A-I , Lipoproteínas de Alta Densidad Pre-beta/biosíntesis , Péptidos/farmacología , Transporte Biológico , Colesterol/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Lipoproteínas de Alta Densidad Pre-beta/efectos de los fármacos , Humanos , Cinética , Imitación MolecularRESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.
Asunto(s)
Endocitosis/fisiología , Receptores de LDL/biosíntesis , Serina Endopeptidasas/fisiología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Línea Celular , LDL-Colesterol/sangre , Regulación hacia Abajo , Humanos , Hígado/metabolismo , Masculino , Ratones , Modelos Biológicos , Proproteína Convertasa 9 , Proproteína Convertasas , Estructura Terciaria de Proteína , Receptores de LDL/fisiología , Serina Endopeptidasas/metabolismoRESUMEN
Adipose tissue appears to be a highly conserved site of cholesteryl ester transfer protein (CETP) expression across species. To investigate the impact of adipose CETP expression on lipid metabolism, we created adipose tissue-specific CETP transgenic (CETPTg) mice. CETP mRNA is predominantly expressed in adipose tissue. Plasma CETP mass and activity are readily detectable in CETPTg mice but not in controls. Plasma lipoprotein analysis shows marked reductions in HDL cholesterol and phospholipids, increases non-HDL lipids, decreases apolipoprotein A-I (apoA-I), and increases apoB. Unexpectedly, CETPTg adipocytes are significantly smaller than those in control mice (44%), triglyceride and cholesterol in adipose tissue were significantly decreased compared with controls (50% and 37%, respectively), and phospholipids showed no significant changes. To study the mechanism, we measured peroxisome proliferator-activated receptor gamma, sterol-regulatory element binding protein-1c, LPL, and hormone-sensitive lipase (HSL) in aP2-CETPTg adipose tissue and controls and found that, except for HSL, all mRNA levels are significantly decreased in the transgenic mice compared with controls (26, 33, and 22%). In conclusion, adipose tissue CETP makes a major contribution to CETP in the circulation, reduces HDL, and increases non-HDL cholesterol levels. Moreover, adipose tissue CETP expression changes triglyceride and cholesterol content and the size of adipocytes.
Asunto(s)
Tejido Adiposo/metabolismo , Proteínas Portadoras/fisiología , Perfilación de la Expresión Génica , Glicoproteínas/fisiología , Lipoproteínas/sangre , Adipocitos/metabolismo , Tejido Adiposo/citología , Tejido Adiposo/efectos de los fármacos , Animales , Apolipoproteínas/análisis , Apolipoproteínas/sangre , Northern Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Riñón/metabolismo , Lípidos/análisis , Lípidos/sangre , Lipoproteínas/metabolismo , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacocinética , Lipoproteínas LDL/metabolismo , Hígado/metabolismo , Ratones , Ratones Transgénicos , Miocardio/metabolismo , PPAR gamma/genética , Bazo/metabolismo , Esterol Esterasa/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genéticaRESUMEN
Hypercholesterolemia is a major risk factor for coronary artery disease. Oxysterols are known to inhibit cholesterol biosynthesis and have been explored as potential antihypercholesterolemic agents. The ability of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) to lower non-HDL cholesterol has been demonstrated in rodent and primate models, but the mechanisms of action remain poorly understood. Here we show in a coactivator recruitment assay and cotransfection assays that the 15-ketosterol is a partial agonist for liver X receptor-alpha and -beta (LXRalpha and LXRbeta). The binding affinity for the LXRs was comparable to those of native oxysterols. In a macrophage cell line of human origin, the 15-ketosterol elevated ATP binding cassette transporter ABCA1 mRNA in a concentration-dependent fashion with a potency similar to those of other oxysterols. We further found that in human embryonic kidney HEK 293 cells, the 15-ketosterol suppressed sterol-responsive element binding protein processing activity and thus inhibited mRNA expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, LDL receptor, and PCSK9. Our data thus provide a molecular basis for the hypocholesterolemic activity of the 15-ketosterol and further suggest its potential antiatherosclerotic benefit as an LXR agonist.
Asunto(s)
Colestenonas/farmacología , Proteínas de Unión al ADN/metabolismo , Hidroximetilglutaril-CoA Reductasas/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/antagonistas & inhibidores , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Células Cultivadas , Proteínas de Unión al ADN/agonistas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Receptores X del Hígado , Receptores Nucleares Huérfanos , Proproteína Convertasa 9 , Proproteína Convertasas , Receptores Citoplasmáticos y Nucleares/agonistas , Serina Endopeptidasas/biosíntesisRESUMEN
Liver X receptors (LXRs) regulate target genes that are critical in lipoprotein metabolism and atherosclerosis. Apolipoprotein AIV (ApoAIV) is an apolipoprotein that is associated with chylomicrons and high-density lipoproteins. Plasma ApoAIV level in humans is inversely correlated with coronary artery events and overexpression of ApoAIV in mice results in significant reduction in atherosclerosis. We report here that LXRs directly regulate apoAIV at the transcriptional level. Treatment of C57B6 mice with a synthetic LXR agonist, T0901317, resulted in significant increases in plasma apoAIV that was associated with high-density lipoprotein. Examination of both intestinal and liver apoAIV mRNA revealed specific increases in liver mRNA only. In a human heptoma HepG2 cell model, apoAIV mRNA was up-regulated upon the treatment with either native or synthetic LXR agonists. Nuclear run-on study revealed a significant increase in the ApoAIV transcriptional rate upon LXR activation. Examination of the human apoAIV proximal promoter revealed a potential LXR response element that demonstrated binding with HepG2 nuclear extracts. Cotransfection studies in HepG2 cells indicated that this responsive element was functional in mediating the human ApoAIV gene response to LXR agonists. In addition, we identified a functional LXR-responsive element at 3' end enhancer region of mouse ApoAIV gene. We conclude that ApoAIV is a direct target gene of LXRs that may contribute to the antiatherogenic effect of LXR activation.
Asunto(s)
Apolipoproteínas A/genética , Arteriosclerosis/tratamiento farmacológico , Arteriosclerosis/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Anticolesterolemiantes/farmacología , Apolipoproteínas A/sangre , Arteriosclerosis/sangre , Arteriosclerosis/genética , Secuencia de Bases , Línea Celular Tumoral , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Humanos , Hidrocarburos Fluorados , Ligandos , Hígado/metabolismo , Receptores X del Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Receptores Nucleares Huérfanos , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Elementos de Respuesta/genética , Receptores X Retinoide/metabolismo , Alineación de Secuencia , Sulfonamidas , Transcripción Genética/genéticaRESUMEN
Liver X receptors (LXRs) are master transcription factors regulating cholesterol and fatty acid metabolism. Treatment of C57B6 mice with a specific synthetic LXR agonist, N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide (T0901317), resulted in elevated high-density lipoprotein (HDL) cholesterol as well as plasma and liver triglycerides. Peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists are known to induce peroxisomal fatty acid beta-oxidation and also mediate HDL cholesterol metabolism. We have explored the hypothesis that simultaneous activation of PPARalpha and LXR may lead to additive effects on HDL cholesterol elevation as well as attenuation of triglyceride accumulation. Coadministration of T0901317 and the specific PPARalpha agonist [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (Wy14643)] in mice led to synergistic elevation of HDL cholesterol that was primarily associated with enlarged HDL particles enriched with apoE and apoAI. Liver phospholipid transfer protein (PLTP) mRNA and plasma PLTP activity were additively elevated, suggesting a role of PLTP in the observed HDL cholesterol elevation. Moderate increases in plasma triglyceride levels induced by LXR activation was reduced, whereas the accumulation of triglyceride in the liver was not altered upon coadministration of the PPARalpha agonist. Peroxisomal fatty acid beta-oxidation in the liver was dramatically elevated upon PPARalpha activation as expected. Interestingly, activation of LXRs via T0901317 also led to a significant increase in peroxisomal fatty acid beta-oxidation. Sterol regulatory element binding protein 1c expression was dramatically up-regulated by the LXR agonist but was not changed with PPARalpha agonist treatment. Liver lipoprotein lipase expression was additively increased upon LXR agonist and PPARalpha agonist coadministration. Our studies mark the first exploration of nuclear receptor interplay on lipid homeostasis in vivo.
Asunto(s)
Anticolesterolemiantes/farmacología , Lipoproteínas HDL/metabolismo , Hígado/efectos de los fármacos , Proteínas de Transferencia de Fosfolípidos , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/agonistas , Triglicéridos/metabolismo , Animales , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN , Hidrocarburos Fluorados , Hígado/metabolismo , Receptores X del Hígado , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores Nucleares Huérfanos , Pirimidinas/farmacología , SulfonamidasRESUMEN
Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells). Examination of primary mouse astrocytes also revealed significant induction of apoE mRNA, and protein expression and secretion following incubation with LXR/RXR agonists. Moreover, treatment of mice with a specific synthetic LXR agonist T0901317 resulted in up-regulation of apoE mRNA and protein in both hippocampus and cerebral cortex, indicating that apoE expression in brain can be up-regulated by LXR agonists in vivo. Along with a dramatic induction of ABCA1 cholesterol transporter expression, these ligands effectively mediate cholesterol efflux in both CCF-STTG1 cells and mouse astrocytes in the presence or absence of apolipoprotein AI (apoAI). Our studies provide strong evidence that small molecule LXR/RXR agonists can effectively mediate apoE synthesis and secretion as well as cholesterol homeostasis in astrocytes. LXR/RXR agonists may have significant impact on the pathogenesis of multiple neurological diseases, including AD.
Asunto(s)
Apolipoproteínas E/biosíntesis , Colesterol/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Ácido Retinoico/fisiología , Factores de Transcripción/fisiología , Animales , Anticolesterolemiantes/farmacología , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Línea Celular Tumoral , Células Cultivadas , Proteínas de Unión al ADN , Dimerización , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Humanos , Hidrocarburos Fluorados , Receptores X del Hígado , Ratones , Ratones Endogámicos C57BL , Receptores Nucleares Huérfanos , Receptores X Retinoide , SulfonamidasRESUMEN
The factors involved in the generation of larger high density lipoprotein (HDL) particles, HDL1 and HDLc, are still not well understood. Administration of a specific synthetic liver X receptor (LXR) agonist, T0901317, in mice resulted in an increase of not only HDL cholesterol but also HDL particle size (Cao, G., Beyer, T. P., Yang, X. P., Schmidt, R. J., Zhang, Y., Bensch, W. R., Kauffman, R. F., Gao, H., Ryan, T. P., Liang, Y., Eacho, P. I., and Jiang, X. C. (2002) J. Biol. Chem. 277, 39561-39565). We have investigated the roles that apoE and CETP may play in this process. We treated apoE-deficient, cholesterol ester transport protein (CETP) transgenic, and wild type mice with various doses of the LXR agonist and monitored their HDL levels. Fast protein liquid chromatography and apolipoprotein analysis revealed that in apoE knockout mouse plasma, there was neither induction of larger HDL formation nor increase of HDL cholesterol, suggesting that apoE is essential for the LXR agonist effects on HDL metabolism. In CETP transgenic mice, CETP expression completely abolished LXR agonist-mediated HDL enlargement and greatly attenuated HDL cholesterol levels. Analysis of HDL particles by electron microscope and nondenaturing gel electrophoresis revealed similar findings. In apoE-deficient mice, LXR agonist also produced a significant increase in very low density lipoprotein/low density lipoprotein cholesterol and apolipoprotein B content. Our studies provide direct evidence that apoE and CETP are intimately involved in the accumulation of the enlarged HDL (HDL1 or HDLc) particles in mice.
Asunto(s)
Apolipoproteínas E/metabolismo , Proteínas Portadoras/metabolismo , Glicoproteínas , Lipoproteínas HDL/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Proteínas de Transferencia de Ésteres de Colesterol , Electroforesis en Gel de Poliacrilamida , Lipoproteínas HDL/química , Ratones , Ratones Endogámicos C57BL , Microscopía ElectrónicaRESUMEN
Natural products have been identified as ligands for a number of members of the nuclear hormone receptor (NHR) superfamily. Often these natural products are used as dietary supplements to treat myriad ailments ranging from perimenopausal hot flashes to hypercholesterolemia and reduced cognitive function. Examples of some natural product ligands for NHRs include genestein (estrogen receptors NR3A1 and NR3A2), guggulsterone (farnesoid X receptor NR1H4), and St. John's wort (pregnane X receptor, NR1I2). In this study, we identified the first nonoxysterol natural product that functions as a ligand for the liver X receptor (LXRalpha and LXRbeta; NR1H3, NR1H2), a NHR that acts as the receptor for oxysterols and plays a key role in regulation of cholesterol metabolism and transport as well as glucose metabolism. We show that paxilline, a fungal metabolite, is an efficacious agonist of both LXRalpha and LXRbeta in biochemical and in vitro cell-based assays. Paxilline binds directly to both receptors and is an activator of LXR-dependent transcription in cell-based reporter assays. We also demonstrate that paxilline binding to the receptors results in efficient activation of transcription of two physiological LXR target genes, ABCA1 and SREBP. The discovery of paxilline, the first reported nonoxysterol natural product ligand of the LXRs, may provide insight into the mechanism of ligand recognition by these receptors and reaffirms the utility of examining natural product libraries for identifying novel NHR ligands.