RESUMEN
The acyltransferase LCAT mediates FA esterification of plasma cholesterol. In vitro studies have shown that LCAT also FA-esterifies several oxysterols, but in vivo evidence is lacking. Here, we measured both free and FA-esterified forms of sterols in 206 healthy volunteers and 8 individuals with genetic LCAT deficiency, including familial LCAT deficiency (FLD) and fish-eye disease (FED). In the healthy volunteers, the mean values of the ester-to-total molar ratios of the following sterols varied: 4ß-hydroxycholesterol (4ßHC), 0.38; 5,6α-epoxycholesterol (5,6αEC), 0.46; 5,6ß-epoxycholesterol (5,6ßEC), 0.51; cholesterol, 0.70; cholestane-3ß,5α,6ß-triol (CT), 0.70; 7-ketocholesterol (7KC), 0.75; 24S-hydroxycholesterol (24SHC), 0.80; 25-hydroxycholesterol (25HC), 0.81; 27-hydroxycholesterol (27HC), 0.86; and 7α-hydroxycholesterol (7αHC), 0.89. In the individuals with LCAT deficiency, the plasma levels of the FA-esterified forms of cholesterol, 5,6αEC, 5,6ßEC, CT, 7αHC, 7KC, 24SHC, 25HC, and 27HC, were significantly lower than those in the healthy volunteers. The individuals with FLD had significantly lower FA-esterified forms of 7αHC, 24SHC, and 27HC than those with FED. It is of note that, even in the three FLD individuals with negligible plasma cholesteryl ester, substantial amounts of the FA-esterified forms of 4ßHC, 5,6αEC, 7αHC, 7KC, and 27HC were present. We conclude that LCAT has a major role in the FA esterification of many plasma oxysterols but contributes little to the FA esterification of 4ßHC. Substantial FA esterification of 4ßHC, 5,6αEC, 7αHC, 7KC, and 27HC is independent of LCAT.
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Hidroxicolesteroles/sangre , Hidroxicolesteroles/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Adulto , Estudios de Casos y Controles , Esterificación , Femenino , Humanos , Masculino , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Adulto JovenRESUMEN
BACKGROUND: The risk of cardiovascular disease and mortality in salt-sensitive patients with diabetes mellitus and uncontrolled nocturnal hypertension is high. The SACRA (Sodium-Glucose Cotransporter 2 [SGLT2] Inhibitor and Angiotensin Receptor Blocker [ARB] Combination Therapy in Patients With Diabetes and Uncontrolled Nocturnal Hypertension) study investigated changes in blood pressure (BP) with empagliflozin plus existing antihypertensive therapy. METHODS: This multicenter, double-blind, parallel study was conducted in Japan. Adult patients with type 2 diabetes mellitus and uncontrolled nocturnal hypertension receiving stable antihypertensive therapy including angiotensin receptor blockers were randomized to 12 weeks' treatment with empagliflozin 10 mg once daily or placebo. Clinic BP was measured at baseline and weeks 4, 8, and 12; 24-hour ambulatory BP monitoring was performed at baseline and week 12; and morning home BP was determined for 5 days before each visit. The primary efficacy end point was change from baseline in nighttime BP (ambulatory BP monitoring). RESULTS: One hundred thirty-two nonobese, older patients with well-controlled blood glucose were randomized (mean age 70 years, mean body mass index 26 kg/m2). Empagliflozin, but not placebo, significantly reduced nighttime systolic BP versus baseline (-6.3 mm Hg; P=0.004); between-group difference in change from baseline was -4.3 mm Hg (P=0.159). Reductions in daytime, 24-hour, morning home, and clinic systolic BP at 12 weeks with empagliflozin were significantly greater than with placebo (-9.5, -7.7, -7.5, and -8.6 mm Hg, respectively; all P≤0.002). Between-group differences in body weight and glycosylated hemoglobin reductions were significant, but small (-1.3 kg and -0.33%; both P<0.001). At 4 weeks, N-terminal pro-B-type natriuretic peptide levels were reduced to a greater extent in the empagliflozin versus placebo group (-12.1%; P=0.013); atrial natriuretic peptide levels decreased with empagliflozin versus placebo at weeks 4 and 12 (-8.2% [P=0.008] and -9.7% [P=0.019]). Changes in antihypertensive medication during the study did not differ significantly between groups. CONCLUSIONS: Nonseverely obese older diabetes patients with uncontrolled nocturnal hypertension showed significant BP reductions without marked reductions in glucose with the addition of empagliflozin to existing antihypertensive and antidiabetic therapy. Use of sodium-glucose cotransporter 2 inhibitors in specific groups (eg, those with nocturnal hypertension, diabetes, and high salt sensitivity) could help reduce the risk of heart failure and cardiovascular mortality. CLINICAL TRIAL REGISTRATION: URL: https://www. CLINICALTRIALS: gov. Unique identifier: NCT03050229.
RESUMEN
Objective- Inhibition of HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is atheroprotective primarily by decreasing plasma LDL (low-density lipoprotein)-cholesterol. However, it is unknown whether inhibition of HMGCR in myeloid cells contributes to this atheroprotection. We sought to determine the role of myeloid HMGCR in the development of atherosclerosis. Approach and Results- We generated mice with genetically reduced Hmgcr in myeloid cells ( Hmgcr m-/m-) using LysM (Cre) and compared various functions of their macrophages to those of Hmgcr fl/fl control mice. We further compared the extent of atherosclerosis in Hmgcr m-/ m- and Hmgcr fl/fl mice in the absence of Ldlr (LDL receptor). Hmgcr m-/ m- macrophages and granulocytes had significantly lower Hmgcr mRNA expression and cholesterol biosynthesis than Hmgcr fl/fl cells. In vitro, Hmgcr m-/ m- monocytes/macrophages had reduced ability to migrate, proliferate, and survive compared with Hmgcr fl/fl monocytes/macrophages. However, there was no difference in ability to adhere, phagocytose, store lipids, or polarize to M1 macrophages between the 2 types of macrophages. The amounts of plasma membrane-associated small GTPase proteins, such as RhoA (RAS homolog family member A), were increased in Hmgcr m-/ m- macrophages. In the setting of Ldlr deficiency, Hmgcr m-/ m- mice developed significantly smaller atherosclerotic lesions than Hmgcr fl/fl mice. However, there were no differences between the 2 types of mice either in plasma lipoprotein profiles or in the numbers of proliferating or apoptotic cells in the lesions in vivo. The in vivo migration of Hmgcr m-/ m- macrophages to the lesions was reduced compared with Hmgcr fl/fl macrophages. Conclusions- Genetic reduction of HMGCR in myeloid cells may exert atheroprotective effects primarily by decreasing the migratory activity of monocytes/macrophages to the lesions.
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Aorta/enzimología , Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , Movimiento Celular , Hidroximetilglutaril-CoA Reductasas/metabolismo , Macrófagos Peritoneales/enzimología , Monocitos/enzimología , Traslado Adoptivo , Animales , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Hidroximetilglutaril-CoA Reductasas/genética , Lípidos/sangre , Macrófagos Peritoneales/patología , Macrófagos Peritoneales/trasplante , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/patología , Proteínas de Unión al GTP Monoméricas/metabolismo , Fenotipo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de SeñalRESUMEN
Matrix-free laser desorption ionization (LDI) is a rapid and versatile technique for the ionization of small, UV-light-absorbing molecules. Indeed, many natural products such as polyphenols exhibit inherent LDI properties, potentially facilitating their detection from highly complex samples such as crude extracts. With this in mind, the present work thoroughly evaluated the potential of LDI as an analytical tool for the chemical profiling and differentiation of propolis samples obtained from different global regions. Propolis is a complex bee product containing, among others, significant amounts of phenolic constituents that may show LDI effects. The present work will demonstrate that LDI not only provides reproducible and highly specific fingerprint spectra for each of the tested samples, it further allows their clear differentiation by principal compound analysis (PCA). Contrary to classical analytical approaches such as LC- or GC-MS, LDI does not require time-consuming sample preparation and method optimization procedures. Thus, the technique represents a most interesting analytical tool and potent supplement to classic LC-MS for quality control of herbal pharmaceuticals and dietary supplements. Present results clearly support this approach and further suggest the use of LDI as a versatile tool for the automated analysis of large sample batches on an industrial scale. Graphical abstract á .
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Fenoles/análisis , Própolis/química , Control de Calidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Cromatografía Líquida de Alta Presión/métodos , Análisis de Componente Principal , Reproducibilidad de los ResultadosRESUMEN
Alkaloids represent a group of biologically most interesting compounds commonly used in modern medicines but also known for exhibiting severe toxic effects. Therefore, the detection of alkaloids is an important issue in quality control of plants, dietary supplements, and herbal pharmaceutical and mostly facilitated by methods such as GC or LC-MS. However, benefitting from the development of selective matrices as well as requiring very little sample preparation, MALDI-MS may also provide a valuable supplement to these standard analytical methods. With this in mind, the present study highlights recent advances in the development of bithiophenic matrix molecules designed for the selective detection of alkaloids. Overall four new bithiophenic matrix molecules (BMs) were tested on different analytes belonging to various chemical families such as alkaloids, curcuminoids, benzopyrones, flavonoids, steroids, and peptides (I). All BMs were further compared to the commercial matrices α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) in terms of their signal response as well as their matrix noise formation (II). Based on these results the most promising candidate, 3-(5'-pentafluorophenylmethylsulfanyl-[2,2']bithiophenyl-5-ylsulfanyl)propionitrile (PFPT3P), was tested on highly complex samples such as the crude extracts of Colchicum autumnale, RYTMOPASC ® solution (a herbal pharmaceutical containing sparteine and rubijervine), as well as strychnine-spiked human plasma (III). For the latter, an evaluation of the limit of detection was performed. Eventually, a simplified protocol for the direct MALDI detection of major alkaloids from pulverized plant material of Atropa belladonna and Senecio vulgaris is presented (IV). Graphical abstract Selective MALDI MATRICES for Alkaloid Detection.
Asunto(s)
Alcaloides/análisis , Técnicas de Química Analítica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Atropa belladonna/química , Colchicum/química , Suplementos Dietéticos/análisis , Suplementos Dietéticos/normas , Límite de Detección , Fenoles/análisis , Compuestos de Sulfhidrilo/análisisRESUMEN
Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.
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Colesterol/sangre , Farnesil Difosfato Farnesil Transferasa/genética , Hígado/enzimología , Animales , Vías Biosintéticas , Colesterol/biosíntesis , Farnesil Difosfato Farnesil Transferasa/metabolismo , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hígado/fisiopatología , Masculino , Ratones TransgénicosRESUMEN
Hydrolysis of intracellular cholesteryl ester (CE) is the rate-limiting step in the efflux of cholesterol from macrophage foam cells. In mouse peritoneal macrophages (MPMs), this process is thought to involve several enzymes: hormone-sensitive lipase (Lipe), carboxylesterase 3 (Ces3), neutral CE hydrolase 1 (Nceh1). However, there is some disagreement over the relative contributions of these enzymes. To solve this problem, we first compared the abilities of several compounds to inhibit the hydrolysis of CE in cells overexpressing Lipe, Ces3, or Nceh1. Cells overexpressing Ces3 had negligible neutral CE hydrolase activity. We next examined the effects of these inhibitors on the hydrolysis of CE and subsequent cholesterol trafficking in MPMs. CE accumulation was increased by a selective inhibitor of Nceh1, paraoxon, and two nonselective inhibitors of Nceh1, (+)-AS115 and (-)-AS115, but not by two Lipe-selective inhibitors, orlistat and 76-0079. Paraoxon inhibited cholesterol efflux to apoA-I or HDL, while 76-0079 did not. These results suggest that Nceh1 plays a dominant role over Lipe in the hydrolysis of CE and subsequent cholesterol efflux in MPMs.
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Ésteres del Colesterol/metabolismo , Macrófagos Peritoneales/enzimología , Esterol Esterasa/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/genética , Carboxilesterasa/genética , Carboxilesterasa/metabolismo , Ésteres del Colesterol/genética , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Hidrólisis , Ratones , Ratones Noqueados , Esterol Esterasa/antagonistas & inhibidores , Esterol Esterasa/genéticaRESUMEN
An excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis.
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Apoptosis , Estrés del Retículo Endoplásmico , Hidroxicolesteroles/metabolismo , Macrófagos Peritoneales/enzimología , Transducción de Señal , Esterol Esterasa/metabolismo , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Animales , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/patología , Macrófagos Peritoneales/patología , Ratones , Ratones Noqueados , Esterol Esterasa/genética , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismoRESUMEN
Insulin resistance is often associated with obesity and can precipitate type 2 diabetes. To date, most known approaches that improve insulin resistance must be preceded by the amelioration of obesity and hepatosteatosis. Here, we show that this provision is not mandatory; insulin resistance and hyperglycemia are improved by the modification of hepatic fatty acid composition, even in the presence of persistent obesity and hepatosteatosis. Mice deficient for Elovl6, the gene encoding the elongase that catalyzes the conversion of palmitate to stearate, were generated and shown to become obese and develop hepatosteatosis when fed a high-fat diet or mated to leptin-deficient ob/ob mice. However, they showed marked protection from hyperinsulinemia, hyperglycemia and hyperleptinemia. Amelioration of insulin resistance was associated with restoration of hepatic insulin receptor substrate-2 and suppression of hepatic protein kinase C epsilon activity resulting in restoration of Akt phosphorylation. Collectively, these data show that hepatic fatty acid composition is a new determinant for insulin sensitivity that acts independently of cellular energy balance and stress. Inhibition of this elongase could be a new therapeutic approach for ameliorating insulin resistance, diabetes and cardiovascular risks, even in the presence of a continuing state of obesity.
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Acetiltransferasas/metabolismo , Dieta Aterogénica , Grasas de la Dieta/farmacología , Resistencia a la Insulina , Obesidad/metabolismo , Acetiltransferasas/deficiencia , Acetiltransferasas/genética , Animales , Peso Corporal/efectos de los fármacos , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Grasas de la Dieta/administración & dosificación , Elongasas de Ácidos Grasos , Eliminación de Gen , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular/fisiología , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Noqueados , Obesidad/inducido químicamente , Obesidad/genética , Fosfoproteínas/fisiología , Fosforilación , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
The role of macrophage lipoprotein lipase (LpL) in the development of atherosclerosis and adiposity was examined in macrophage LpL knockout (MLpLKO) mice. MLpLKO mice were generated using cre-loxP gene targeting. Loss of LpL in macrophages did not alter plasma LpL activity or lipoprotein levels. Incubation of apolipoprotein E (ApoE)-deficient ß-VLDL with peritoneal macrophages from ApoE knockout mice lacking macrophage LpL (MLpLKO/ApoEKO) led to less cholesteryl ester formation than that found with ApoEKO macrophages. MLpLKO/ApoEKO macrophages had reduced intracellular triglyceride levels, with decreased CD36 and carnitine palmitoyltransferase-1 mRNA levels compared with ApoEKO macrophages, when incubated with VLDL. Although both MLpLKO/ApoEKO and ApoEKO mice developed comparable hypercholesterolemia in response to feeding with a Western-type diet for 12 weeks, atherosclerosis was less in MLpLKO/ApoEKO mice. Epididymal fat mass and gene expression levels associated with inflammation did not differ between the two groups. In conclusion, macrophage LpL plays an important role in the development of atherosclerosis but not adiposity.
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Adiposidad/fisiología , Aterosclerosis/enzimología , Lipoproteína Lipasa/metabolismo , Macrófagos Peritoneales/enzimología , Macrófagos/enzimología , Adiposidad/genética , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/patología , Northern Blotting , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Lipoproteína Lipasa/genética , Ratones , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
OBJECTIVE: 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) catalyzes the rate-limiting step in cholesterol biosynthesis and has proven to be an effective target of lipid-lowering drugs, statins. The aim of this study was to understand the role of hepatic HMGCR in vivo. METHODS AND RESULTS: To disrupt the HMGCR gene in liver, we generated mice homozygous for a floxed HMGCR allele and heterozygous for a transgene encoding Cre recombinase under the control of the albumin promoter (liver-specific HMGCR knockout mice). Ninety-six percent of male and 71% of female mice died by 6 weeks of age, probably as a result of liver failure or hypoglycemia. At 5 weeks of age, liver-specific HMGCR knockout mice showed severe hepatic steatosis with apoptotic cells, hypercholesterolemia, and hypoglycemia. The hepatic steatosis and death were completely reversed by providing the animals with mevalonate, indicating its essential role in normal liver function. There was a modest decrease in hepatic cholesterol synthesis in liver-specific HMGCR knockout mice. Instead, they showed a robust increase in the fatty acid synthesis, independent of sterol regulatory element binding protein-1c. CONCLUSIONS: Hepatocyte HMGCR is essential for the survival of mice, and its abrogation elicits hepatic steatosis with jaundice and hypoglycemia.
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Hígado Graso/etiología , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes/fisiología , Hígado/enzimología , Animales , Femenino , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes/genética , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/análisis , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genéticaRESUMEN
Postprandial hyperglycemia and/or hyperlipidemia can contribute to development of atherosclerosis in patients with type 2 diabetes mellitus (T2DM). The objective of this study was to compare the effects of miglitol and sitagliptin on postprandial glucose and lipid metabolism in patients with T2DM. Thirty-five patients with T2DM were randomized to 2 groups receiving miglitol (150 mg/day) or sitagliptin (50 mg/day). Serum variables related to glucose and lipid metabolism were measured before and after treatment for 10 weeks and at 0, 60, and 120 min using a cookie-loading test (CLT). After 10 weeks of treatment, miglitol (n = 16) and sitagliptin (n = 18) caused a similarly significant decrease in hemoglobin A1c (mean: 7.6% to 7.3% versus 8.0% to 7.6%) and a significant increase in fasting insulin levels, with a greater increase observed in the miglitol group than in the sitagliptin group (p=0.03). In addition, a significant decrease in the change in glucose levels after the CLT was observed in both groups, with a greater decrease observed in the miglitol group than in the sitagliptin group (p=0.02). The miglitol group also showed a greater decrease in the change in insulin levels after the CLT than the sitagliptin group (p<0.01). The lipid and lipoprotein levels did not show any significant differences between the groups after the CLT. Our results suggested that miglitol and sitagliptin treatment resulted in similar glycemic control but that a greater decrease in postprandial glucose and insulin levels was observed with miglitol compared with sitagliptin in patients with T2DM.
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1-Desoxinojirimicina/análogos & derivados , Glucemia/efectos de los fármacos , Diabetes Mellitus Tipo 2/metabolismo , Hipoglucemiantes/farmacología , Lipoproteínas/metabolismo , Pirazinas/farmacología , Triazoles/farmacología , 1-Desoxinojirimicina/farmacología , 1-Desoxinojirimicina/uso terapéutico , Anciano , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Femenino , Humanos , Hipoglucemiantes/uso terapéutico , Insulina/sangre , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Periodo Posprandial/efectos de los fármacos , Pirazinas/uso terapéutico , Fosfato de Sitagliptina , Triazoles/uso terapéuticoRESUMEN
Overexpression of sterol regulatory element-binding protein-1c (SREBP-1c) in ß cells causes impaired insulin secretion and ß cell dysfunction associated with diminished pancreatic duodenal homeodomain transcription factor-1 (PDX-1) expression in vitro and in vivo. To identify the molecular mechanism responsible for this effect, the mouse Pdx-1 gene promoter (2.7 kb) was analyzed in ß cell and non-ß cell lines. Despite no apparent sterol regulatory element-binding protein-binding sites, the Pdx-1 promoter was suppressed by SREBP-1c in ß cells in a dose-dependent manner. PDX-1 activated its own promoter. The E-box (-104/-99 bp) in the proximal region, occupied by ubiquitously expressed upstream stimulatory factors (USFs), was crucial for the PDX-1-positive autoregulatory loop through direct PDX-1·USF binding. This positive feedback activation was a prerequisite for SREBP-1c suppression of the promoter in non-ß cells. SREBP-1c and PDX-1 directly interact through basic helix-loop-helix and homeobox domains, respectively. This robust SREBP-1c·PDX-1 complex interferes with PDX-1·USF formation and inhibits the recruitment of PDX-1 coactivators. SREBP-1c also inhibits PDX-1 binding to the previously described PDX-1-binding site (-2721/-2646 bp) in the distal enhancer region of the Pdx-1 promoter. Endogenous up-regulation of SREBP-1c in INS-1 cells through the activation of liver X receptor and retinoid X receptor by 9-cis-retinoic acid and 22-hydroxycholesterol inhibited PDX-1 mRNA and protein expression. Conversely, SREBP-1c RNAi restored Pdx-1 mRNA and protein levels. Through these multiple mechanisms, SREBP-1c, when induced in a lipotoxic state, repressed PDX-1 expression contributing to the inhibition of insulin expression and ß cell dysfunction.
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Proteínas de Homeodominio/biosíntesis , Células Secretoras de Insulina/metabolismo , Insulina/biosíntesis , Elementos de Respuesta/fisiología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Transactivadores/biosíntesis , Regulación hacia Arriba/fisiología , Alitretinoína , Animales , Antineoplásicos/farmacología , Células Hep G2 , Proteínas de Homeodominio/genética , Humanos , Insulina/genética , Receptores X del Hígado , Ratones , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Transactivadores/genética , Tretinoina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Factores Estimuladores hacia 5'/genética , Factores Estimuladores hacia 5'/metabolismoRESUMEN
RATIONALE: Hydrolysis of intracellular cholesterol ester (CE) is the key step in the reverse cholesterol transport in macrophage foam cells. We have recently shown that neutral cholesterol ester hydrolase (Nceh)1 and hormone-sensitive lipase (Lipe) are key regulators of this process in mouse macrophages. However, it remains unknown which enzyme is critical in human macrophages and atherosclerosis. OBJECTIVE: We aimed to identify the enzyme responsible for the CE hydrolysis in human macrophages and to determine its expression in human atherosclerosis. METHODS AND RESULTS: We compared the expression of NCEH1, LIPE, and cholesterol ester hydrolase (CES1) in human monocyte-derived macrophages (HMMs) and examined the effects of inhibition or overexpression of each enzyme in the cholesterol trafficking. The pattern of expression of NCEH1 was similar to that of neutral CE hydrolase activity during the differentiation of HMMs. Overexpression of human NCEH1 increased the hydrolysis of CE, thereby stimulating cholesterol mobilization from THP-1 macrophages. Knockdown of NCEH1 specifically reduced the neutral CE hydrolase activity. Pharmacological inhibition of NCEH1 also increased the cellular CE in HMMs. In contrast, LIPE was barely detectable in HMMs, and its inhibition did not decrease neutral CE hydrolase activity. Neither overexpression nor knockdown of CES1 affected the neutral CE hydrolase activity. NCEH1 was expressed in CD68-positive macrophage foam cells of human atherosclerotic lesions. CONCLUSIONS: NCEH1 is expressed in human atheromatous lesions, where it plays a critical role in the hydrolysis of CE in human macrophage foam cells, thereby contributing to the initial part of reverse cholesterol transport in human atherosclerosis.
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Proteínas Portadoras/fisiología , Colesterol/metabolismo , Macrófagos/enzimología , Serina Proteasas/fisiología , Esterol Esterasa/fisiología , Anciano , Anciano de 80 o más Años , Aterosclerosis/enzimología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Transporte Biológico/fisiología , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Células Cultivadas , Femenino , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Células HEK293 , Humanos , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/enzimología , Monocitos/metabolismo , Serina Proteasas/biosíntesis , Serina Proteasas/genética , Esterol Esterasa/biosíntesis , Esterol Esterasa/genéticaRESUMEN
To address the effects of ezetimibe on high-density lipoprotein (HDL) metabolism, the HDL subclasses, cholesteryl ester transfer protein (CETP), and lecithin-cholesterol acyltransferase (LCAT) were measured in patients with type 2 diabetes mellitus (T2DM). Twenty-three hypercholesterolemic patients with T2DM were treated with 10 mg of ezetimibe daily for 12 weeks. Plasma total cholesterol (TC), low-density lipoprotein (LDL)-cholesterol (C), HDL-C, HDL(2)-C, HDL(3)-C, CETP mass, and LCAT activity were measured. HDL-C and HDL(2)-C increased by 5% (p<0.05) and 12% (p<0.01), respectively, in response to ezetimibe. Of the 23 patients, 21 had decreased CETP mass, which led to an average reduction of 20% (p<0.0001). LCAT activity also decreased by 6% (p<0.01). A significant positive correlation was found in the changes from baseline between HDL(2)-C and CETP mass, whereas a significant inverse relationship was observed between HDL(3)-C and CETP mass. Furthermore, the change in HDL-C was positively correlated with the change in LCAT activity. In conclusion, ezetimibe may affect HDL metabolism and reverse cholesterol transport, especially CETP, in T2DM. These observations may provide some insights into how ezetimibe prevents atherosclerosis.
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Azetidinas/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas de Transferencia de Ésteres de Colesterol/sangre , Diabetes Mellitus Tipo 2/sangre , Glicoproteínas de Membrana/antagonistas & inhibidores , Anciano , Anticolesterolemiantes/farmacología , Anticolesterolemiantes/uso terapéutico , Azetidinas/uso terapéutico , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , HDL-Colesterol/sangre , HDL-Colesterol/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Ezetimiba , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Masculino , Persona de Mediana Edad , Proteína Niemann-Pick C1 , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismoRESUMEN
Sterol regulatory element-binding protein (SREBP)-1 is a key transcription factor for the regulation of lipogenic enzyme genes in the liver. Polyunsaturated fatty acids (PUFA) selectively suppress hepatic SREBP-1, but molecular mechanisms remain largely unknown. To gain insight into this regulation, we established in vivo reporter assays to assess the activities of Srebf1c transcription and proteolytic processing. Using these in vivo reporter assays, we showed that the primary mechanism for PUFA suppression of SREBP-1 is at the proteolytic processing level and that this suppression in turn decreases the mRNA transcription through lowering SREBP-1 binding to the SREBP-binding element on the promoter ("autoloop regulatory circuit"), although liver X receptor, an activator for Srebf1c transcription, is not involved in this regulation by PUFA. The mechanisms for PUFA suppression of SREBP-1 confirm that the autoloop regulation for transcription is crucial for the nutritional regulation of triglyceride synthesis.
Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Regulación de la Expresión Génica , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Humanos , Hígado/metabolismo , Receptores X del Hígado , Masculino , Ratones , Ratones Endogámicos ICR , Receptores Nucleares Huérfanos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Triglicéridos/metabolismoRESUMEN
We have previously demonstrated that neutral cholesterol ester hydrolase 1 (Nceh1) regulates foam cell formation and atherogenesis through the catalytic activity of cholesterol ester hydrolysis, and that Nceh1 and hormone-sensitive lipase (Lipe) are responsible for the majority of neutral cholesterol ester hydrolase activity in macrophages. There are several cholesterol ester-metabolizing tissues and cells other than macrophages, among which adrenocortical cells are also known to utilize the intracellular cholesterol for steroidogenesis. It has been believed that the mobilization of intracellular cholesterol ester in adrenal glands was facilitated solely by Lipe. We herein demonstrate that Nceh1 is also involved in cholesterol ester hydrolysis in adrenal glands. While Lipe deficiency remarkably reduced the neutral cholesterol ester hydrolase activity in adrenal glands as previously reported, additional inactivation of Nceh1 gene completely abrogated the activity. Adrenal glands were enlarged in proportion to the degree of reduced neutral cholesterol ester hydrolase activity, and the enlargement of adrenal glands and the accumulation of cholesterol esters were most pronounced in the Nceh1/Lipe double-deficient mice. Thus Nceh1 is involved in the adrenal cholesterol metabolism, and the cholesterol ester hydrolytic activity in adrenal glands is associated with the organ enlargement.
Asunto(s)
Glándulas Suprarrenales/anatomía & histología , Colesterol/deficiencia , Serina Proteasas/genética , Esterol Esterasa/genética , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/enzimología , Hormona Adrenocorticotrópica/farmacología , Animales , Expresión Génica , Hidrólisis , Masculino , Ratones , Ratones Mutantes , Tamaño de los Órganos/efectos de los fármacosRESUMEN
Neutral cholesterol ester hydrolase (NCEH) accounts for a large part of the nCEH activity in macrophage foam cells, a hallmark of atherosclerosis, but its subcellular localization and structure-function relationship are unknown. Here, we determined subcellular localization, glycosylation, and nCEH activity of a series of NCEH mutants expressed in macrophages. NCEH is a single-membrane-spanning type II membrane protein comprising three domains: N-terminal, catalytic, and lipid-binding domains. The N-terminal domain serves as a type II signal anchor sequence to recruit NCEH to the endoplasmic reticulum (ER) with its catalytic domain within the lumen. All of the putative N-linked glycosylation sites (Asn(270), Asn(367), and Asn(389)) of NCEH are glycosylated. Glycosylation at Asn(270), which is located closest to the catalytic serine motif, is important for the enzymatic activity. Cholesterol loading by incubation with acetyl-LDL does not change the ER localization of NCEH. In conclusion, NCEH is targeted to the ER of macrophages, where it hydrolyzes CE to deliver cholesterol for efflux out of the cells.
Asunto(s)
Retículo Endoplásmico/metabolismo , Esterol Esterasa/química , Esterol Esterasa/metabolismo , Animales , Biocatálisis , Dominio Catalítico , Bovinos , Línea Celular , Glucosa/metabolismo , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espacio Intracelular/metabolismo , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos C57BL , Transporte de ProteínasRESUMEN
To date, there are very few clinical reports that have compared the effects of ezetimibe on lipid parameters between hypercholesterolemic patients with and without type 2 diabetes mellitus (T2DM). In this study, we recruited patients for hypercholesterolemic groups with T2DM (n = 42; men/women = 24/18; HbA1c = 6.7 ± 5.4%) and without T2DM (n = 21; men/women = 7/14; HbA1c = 5.3 ± 0.4%). Patients were prescribed ezetimibe at a dose of 10 mg/daily for the course of the 12-week study. At baseline and after 12 weeks of treatment, several lipid parameters, including serum low-density-lipoprotein cholesterol (LDL-C), non-high-density-lipoprotein cholesterol (non-HDL-C), high-sensitivity C-reactive protein (hs-CRP), and cholesterol synthesis/absorption-related markers, were measured. Compared with those at the baseline, the levels of LDL-C, non-HDL-C, campesterol, and sitosterol were significantly reduced after 12 weeks of ezetimibe treatment in both groups. After adjusting for confounding factors, such as age, gender, smoking, and BMI, the levels of LDL-C and non-HDL-C displayed significantly greater reductions in the patients with T2DM (-25.1 ± 13.6% in LDL-C, -20.5 ± 11.2% in non-HDL-C) than those without T2DM (-20.5 ± 7.8% in LDL-C, P < 0.05; -17.4 ± 7.6% in non-HDL-C, P < 0.05). The reduction of the level of cholestanol was significantly and positively correlated with those of LDL-C and non-HDL-C in the patients with T2DM. Taken together, these findings indicate that ezetimibe could reduce the levels of atherogenic lipoproteins to a greater extent in hypercholesterolemic patients with T2DM than in those without T2DM.
Asunto(s)
Azetidinas/uso terapéutico , Diabetes Mellitus Tipo 2/complicaciones , Hipercolesterolemia/complicaciones , Hipercolesterolemia/tratamiento farmacológico , Hipolipemiantes/uso terapéutico , Lípidos/sangre , Anciano , Biomarcadores/sangre , Índice de Masa Corporal , Proteína C-Reactiva/análisis , Enfermedades Cardiovasculares/prevención & control , Colestanol/sangre , Colesterol/análogos & derivados , Colesterol/sangre , Colesterol/metabolismo , LDL-Colesterol/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Ezetimiba , Femenino , Humanos , Hipercolesterolemia/sangre , Masculino , Persona de Mediana Edad , Fitosteroles/sangre , Sitoesteroles/sangreRESUMEN
Increased fatty acid (FA) flux and intracellular lipid accumulation (steatosis) give rise to cardiac lipotoxicity in both pathological and physiological conditions. Since hormone-sensitive lipase (HSL) contributes to intracellular lipolysis in adipose tissue and heart, we investigated the impact of HSL disruption on cardiac energy metabolism in response to fasting and refeeding. HSL-knockout (KO) mice and wild-type (WT) littermates were fasted for 24 h, followed by â¼6 h of refeeding. Plasma FA concentration in WT mice was elevated twofold with fasting, whereas KO mice lacked this elevation, resulting in twofold lower cardiac FA uptake compared with WT mice. Echocardiography showed that fractional shortening was 15% decreased during fasting in WT mice and was associated with steatosis, whereas both of these changes were absent in KO mice. Compared with Langendorff-perfused hearts isolated from fasted WT mice, the isolated KO hearts also displayed higher contractile function and a blunted response to FA. Although cardiac glucose uptake in KO mice was comparable with WT mice under all conditions tested, cardiac VLDL uptake and lipoprotein lipase (LPL) activity were twofold higher in KO mice during fasting. The KO hearts showed undetectable activity of neutral cholesteryl esterase and 40% lower non-LPL triglyceride lipase activity compared with WT hearts in refed conditions accompanied by overt steatosis, normal cardiac function, and increased mRNA expression of adipose differentiation-related protein. Thus, the dissociation between cardiac steatosis and functional sequelae observed in HSL-KO mice suggests that excess FA influx, rather than steatosis per se, appears to play an important role in the pathogenesis of cardiac lipotoxicity.