RESUMEN

The present study aims at evaluating a batch scale biosorption potential of Moringa oleifera leaves (MOL) for the removal of Pb(II) from aqueous solutions. The MOL biomass was characterized by FTIR, SEM, EDX, and BET. The impact of initial concentrations of Pb (II), adsorbent dosage, pH, contact time, coexisting inorganic ions (Ca2+, Na+, K+, Mg2+, CO32-, HCO3-, Cl-), electrical conductivity (EC) and total dissolved salts (TDS) in water was investigated. The results revealed that maximum biosorption (45.83 mg/g) was achieved with adsorbent dosage 0.15 g/100 mL while highest removal (98.6%) was obtained at adsorbent biomass 1.0 g/100 mL and pH 6. The presence of coexisting inorganic ions in water showed a decline in Pb(II) removal (8.5% and 5%) depending on the concentrations of ions. The removal of Pb(II) by MOL decreased from 97% to 89% after five biosorption/desorption cycles with 0.3 M HCl solution. Freundlich model yielded a better fit for equilibrium data and the pseudo-second-order well described the kinetics of Pb(II) biosorption. FTIR spectra showed that -OH, C-H, -C-O, -C = O, and -O-C functional groups were involved in the biosorption of Pb(II). The change in Gibbs free energy (ΔG = -28.10 kJ/mol) revealed that the biosorption process was favorable and thermodynamically driven. The results suggest MOL as a low cost, environment-friendly alternative biosorbent for the remediation of Pb(II) contaminated water.

Asunto(s)

Moringa oleifera , Contaminantes Químicos del Agua , Adsorción , Biodegradación Ambiental , Biomasa , Concentración de Iones de Hidrógeno , Cinética , Plomo

RESUMEN

Tumor necrosis factor- (TNF-) α is a proinflammatory proatherogenic cytokine. Infliximab, an anti-TNF-α monoclonal antibody, is effective in treating rheumatoid arthritis. However, its impact on cardiovascular burden and lipid transport is unclear. The present study investigates the effect of TNF-α and infliximab on reverse cholesterol transport (RCT) proteins. Uptake of modified lipoproteins by macrophages in the vasculature leads to atherogenic foam cell formation. RCT is mediated by proteins including ATP binding cassette transporters A1 (ABCA1), G1 (ABCG1), liver X receptor- (LXR-) α, and 27-hydroxylase. RCT counteracts lipid overload by ridding cells of excess cholesterol. THP-1 human monocytes were incubated with either TNF-α alone or TNF-α with infliximab. Expression of proteins involved in cholesterol efflux was analyzed. TNF-α significantly reduced both ABCA1 and LXR-α mRNA (to 68.5 ± 1.59%, P < 0.05, and 41.2 ± 0.25%, P < 0.01, versus control set as 100%, resp.). Infliximab nullified the TNF-α effect. Results were confirmed by Western blot. Infliximab abolished the increase in foam cells induced by TNF-α. TNF-α treatment significantly reduces ABCA1 and LXR-α expression in monocytes, thus bringing about a proatherogenic state. The anti-TNF drug infliximab, commonly used in rheumatology, restored RCT proteins. This is the first report of an atheroprotective effect of infliximab on RCT in monocytes.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/metabolismo , Anticuerpos Monoclonales/farmacología , Aterosclerosis/metabolismo , Colestanotriol 26-Monooxigenasa/metabolismo , Colesterol/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Transportadoras de Casetes de Unión a ATP/genética , Transporte Biológico/efectos de los fármacos , Línea Celular , Humanos , Infliximab , Lipoproteínas LDL/metabolismo , Receptores X del Hígado , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Receptores Nucleares Huérfanos/genética

RESUMEN

The endoplasmic reticulum (ER) forms a network of tubules and sheets that requires homotypic membrane fusion to be maintained. In metazoans, this process is mediated by dynamin-like guanosine triphosphatases (GTPases) called atlastins (ATLs), which are also required to maintain ER morphology. Previous work suggested that the dynamin-like GTPase Sey1p was needed to maintain ER morphology in Saccharomyces cerevisiae. In this paper, we demonstrate that Sey1p, like ATLs, mediates homotypic ER fusion. The absence of Sey1p resulted in the ER undergoing delayed fusion in vivo and proteoliposomes containing purified Sey1p fused in a GTP-dependent manner in vitro. Sey1p could be partially replaced by ATL1 in vivo. Like ATL1, Sey1p underwent GTP-dependent dimerization. We found that the residual ER-ER fusion that occurred in cells lacking Sey1p required the ER SNARE Ufe1p. Collectively, our results show that Sey1p and its homologues function analogously to ATLs in mediating ER fusion. They also indicate that S. cerevisiae has an alternative fusion mechanism that requires ER SNAREs.

Asunto(s)

Retículo Endoplásmico/metabolismo , Fusión de Membrana/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Retículo Endoplásmico/ultraestructura , Proteínas de Unión al GTP/metabolismo , Técnicas de Inactivación de Genes , Proteínas de la Membrana/metabolismo , Proteínas Qa-SNARE/metabolismo , Saccharomyces cerevisiae/ultraestructura

RESUMEN

Cardiovascular safety of cyclooxygenase (COX)-2-selective inhibitors and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) is of worldwide concern. COX-2 inhibitors and NSAIDs act by inhibiting arachidonic acid metabolism to prostaglandins. They confer a cardiovascular hazard manifested as an elevated risk of myocardial infarction. Mechanisms underlying these cardiovascular effects are uncertain. Here we determine whether interference with cytosolic phospholipase A2 (cPLA-2) or COX-2 through pharmacologic blockade or silencing RNA impacts expression of scavenger receptor CD36 and scavenger receptor A, both involved in cholesterol uptake in monocytes and macrophages. THP-1 human monocytes and human peripheral blood mononuclear cells were exposed to celecoxib, a COX-2 selective inhibitor currently in clinical use, and to arachidonyl trifluoromethyl ketone (AACOCF3), an arachidonic acid analog that selectively inhibits cPLA-2. Celecoxib and AACOCF3 each upregulated expression of CD36, but not scavenger receptor A, as determined by quantitative PCR and immunoblotting. Silencing of cPLA-2 or COX-2 had comparable effects to pharmacologic treatments. Oil red O staining revealed a profound increase in foam cell transformation of THP-1 macrophages exposed to either celecoxib or AACOCF3 (both 25 µM), supporting a role for the COX pathway in maintaining macrophage cholesterol homeostasis. Demonstration of disrupted cholesterol balance by AACOCF3 and celecoxib provides further evidence of the possible mechanism by which COX inhibition may promote lipid overload leading to atheromatous lesion formation and increased cardiovascular events.

Asunto(s)

Ácidos Araquidónicos/efectos adversos , Antígenos CD36/biosíntesis , Inhibidores de la Ciclooxigenasa 2/efectos adversos , Ciclooxigenasa 2/metabolismo , Inhibidores Enzimáticos/efectos adversos , Células Espumosas/enzimología , Fosfolipasas A2 Citosólicas/antagonistas & inhibidores , Pirazoles/efectos adversos , Sulfonamidas/efectos adversos , Antígenos CD36/genética , Celecoxib , Técnicas de Cultivo de Célula , Células Cultivadas , Colesterol/metabolismo , Ciclooxigenasa 2/genética , Expresión Génica , Silenciador del Gen , Humanos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Fosfolipasas A2 Citosólicas/genética , Fosfolipasas A2 Citosólicas/metabolismo , Placa Aterosclerótica/inducido químicamente , Placa Aterosclerótica/enzimología , Receptores Depuradores de Clase A/biosíntesis , Receptores Depuradores de Clase A/genética

RESUMEN

Atherosclerotic cardiovascular disease (ASCVD) contributes to morbidity and mortality in systemic lupus erythematosus (SLE). Immunologic derangements may disrupt cholesterol balance in vessel wall monocytes/macrophages and endothelium. We determined whether lupus plasma impacts expression of cholesterol 27-hydroxylase, an anti-atherogenic cholesterol-degrading enzyme that promotes cellular cholesterol efflux, in THP-1 human monocytes and primary human aortic endothelial cells (HAEC). THP-1 monocytes and HAEC were incubated in medium containing SLE patient plasma or apparently healthy control human plasma (CHP). SLE plasma decreased 27-hydroxylase message in THP-1 monocytes by 47 +/- 8% (p < 0.008) and in HAEC by 51 +/- 5.5% (n = 5, p < 0.001). THP-1 macrophages were incubated in 25% lupus plasma or CHP and cholesterol-loaded (50 microg ml(-1) acetylated low density lipoprotein). Lupus plasma more than doubled macrophage foam cell transformation (74 +/- 3% vs. 35 +/- 3% for CHP, n = 3, p < 0.001). Impaired cholesterol homeostasis in SLE provides further evidence of immune involvement in atherogenesis. Strategies to inhibit or reverse arterial cholesterol accumulation may benefit SLE patients.

Asunto(s)

Aterosclerosis/inmunología , Autoinmunidad , Colestanotriol 26-Monooxigenasa/metabolismo , Colesterol/metabolismo , Células Endoteliales/enzimología , Lupus Eritematoso Sistémico/sangre , Monocitos/enzimología , Adolescente , Adulto , Aterosclerosis/sangre , Estudios de Casos y Controles , Células Cultivadas , Colestanotriol 26-Monooxigenasa/genética , Regulación hacia Abajo , Células Endoteliales/inmunología , Femenino , Células Espumosas/enzimología , Regulación Enzimológica de la Expresión Génica , Homeostasis , Humanos , Interferón gamma/antagonistas & inhibidores , Interferón gamma/inmunología , Lipoproteínas LDL/metabolismo , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/inmunología , Monocitos/inmunología , ARN Mensajero/metabolismo , Receptores de Interferón/antagonistas & inhibidores , Receptores de Interferón/inmunología , Factores de Riesgo , Adulto Joven , Receptor de Interferón gamma

RESUMEN

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor that primarily binds and regulates oxidized low-density lipoprotein (LDL). Expression of LOX-1 is regulated by a feed-forward system stimulated by oxidized LDL (oxLDL), a major component of atherosclerosis. LOX-1 is a homodimer with a reactive backbone that can bind to a host of different ligands, including small molecules, and whole cells. LOX-1 is involved in many intercellular, intracellular, and molecular processes that are atherogenic. LOX-1 levels are elevated within atherosclerotic plaques and its expression is induced by proinflammatory cytokines. The ability of LOX-1 to bind many different ligands and control several atherogenic processes makes this receptor a likely vascular disease biomarker as well as an ideal choice for drug therapy aimed at preventing cardiovascular disease.

Asunto(s)

Aterosclerosis/etiología , Lipoproteínas LDL/metabolismo , Receptores Depuradores de Clase E/metabolismo , Animales , Aterosclerosis/tratamiento farmacológico , Modelos Animales de Enfermedad , Humanos , Ratones , Polimorfismo Genético , Ratas , Especies Reactivas de Oxígeno/metabolismo , Receptores Depuradores de Clase E/química , Receptores Depuradores de Clase E/genética

RESUMEN

Atherosclerosis is a chronic progressive disease that is a major contributor to cardiac death. It is characterized by inflammation and cholesterol deposition in the arterial wall. Excess cholesterol accumulation occurs as a result of an imbalance between delivery and removal and leads to formation of lipid-laden foam cells. Removal of cholesterol through a process known as reverse cholesterol transport requires the coordinated functioning of a number of genes including the P450 27-hydroxylase and the adenosine triphosphate-binding cassette transporter A1 (ABCA1). Reverse cholesterol transport is a key defense against atheroma formation. This review discusses the role of inflammatory processes in impeding reverse cholesterol transport. Particular emphasis is placed on the disruption of cholesterol outflow observed in the presence of cyclooxygenase inhibitors in cultured monocytes/macrophages. These inhibitors, which are used clinically to relieve pain and inflammation, have been associated with increased risk of cardiovascular disease and myocardial infarction. We explore the relationship between suppression of reverse cholesterol transport and harmful cardiac effects of coxibs.

Asunto(s)

Aterosclerosis/etiología , Colesterol/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/patología , Transporte Biológico/efectos de los fármacos , Colesterol 7-alfa-Hidroxilasa/metabolismo , Humanos

RESUMEN

Premature atherosclerotic cardiovascular disease (ASCVD) is a common and devastating complication of systemic lupus erythematosus (SLE). It is likely that immunologic derangements contribute to premature ASCVD in these patients, possibly by disrupting homeostatic mechanisms that orchestrate cholesterol balance in monocytes/macrophages in the artery wall. CD36, a macrophage scavenger receptor responsible for recognition and internalization of oxidized lipids, is a major participant in atherosclerotic foam cell formation. We hypothesized that lupus plasma would affect CD36 expression in a pro-atherogenic manner in THP-1 human monocytes and differentiated macrophages. SLE patient plasma markedly stimulated expression of CD36 message in a dose-dependent fashion in THP-1 human monocytes. A 50% volume/volume concentration of plasma derived from SLE patients increased CD36 mRNA by 71 +/- 8% (n = 3, P < 0.001) above 50% normal human plasma. 50% SLE patient plasma increased CD36 mRNA expression to 290 +/- 12% of no-plasma control (n = 3, P < 0.001), compared with only 118 +/- 3.7% of control in the presence of 50% normal human plasma (n = 3, not significant). 50% lupus plasma also upregulated CD36 protein expression by 482.3 +/- 76.2% (n = 4, P < 0.05), whereas the presence of 50% normal human plasma increased the CD36 protein level by only 239.8 +/- 61.9% (n = 4, P < 0.05). To our knowledge, this is the first demonstration that CD36 expression is enhanced by plasma from patients with an autoimmune disorder. Premature atherosclerosis is common in SLE patients. Upregulation of CD36 may contribute to this pathological process by increasing vulnerability to cholesterol overload. Demonstration of disrupted cholesterol homeostasis in this select group of patients provides further evidence of the involvement of the immune system in atherogenesis and may inform us of the role of CD36 in the general atherogenic process. CD36 may provide a novel therapeutic target in the treatment of ASCVD in SLE patients.

Asunto(s)

Aterosclerosis/sangre , Aterosclerosis/inmunología , Autoinmunidad , Antígenos CD36/metabolismo , Lupus Eritematoso Sistémico/sangre , Monocitos/metabolismo , Adulto , Antígenos CD36/genética , Estudios de Casos y Controles , Línea Celular , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

OBJECTIVE: To determine whether methotrexate (MTX) can overcome the atherogenic effects of cyclooxygenase 2 (COX-2) inhibitors and interferon-gamma (IFNgamma), both of which suppress cholesterol efflux protein and promote foam cell transformation in human THP-1 monocyte/macrophages. METHODS: Message and protein levels of the reverse cholesterol transport proteins cholesterol 27-hydroxylase and ATP-binding cassette transporter A1 (ABCA1) in THP-1 cells were evaluated by real-time polymerase chain reaction and immunoblot, respectively. Expression was evaluated in cells incubated in the presence or absence of the COX-2 inhibitor NS398 or IFNgamma, with and without MTX. Foam cell transformation of lipid-laden THP-1 macrophages was detected with oil red O staining and light microscopy. RESULTS: MTX increased 27-hydroxylase message and completely blocked NS398-induced down-regulation of 27-hydroxylase (mean +/- SEM 112.8 +/- 13.1% for NS398 plus MTX versus 71.1 +/- 4.3% for NS398 alone; P < 0.01). MTX also negated COX-2 inhibitor-mediated down-regulation of ABCA1. The ability of MTX to reverse inhibitory effects on 27-hydroxylase and ABCA1 was blocked by the adenosine A2A receptor-specific antagonist ZM241385. MTX also prevented NS398 and IFNgamma from increasing transformation of lipid-laden THP-1 macrophages into foam cells. CONCLUSION: This study provides evidence supporting the notion of an atheroprotective effect of MTX. Through adenosine A2A receptor activation, MTX promotes reverse cholesterol transport and limits foam cell formation in THP-1 macrophages. This is the first reported evidence that any commonly used medication can increase expression of antiatherogenic reverse cholesterol transport proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular disease in rheumatoid arthritis patients is through facilitation of cholesterol outflow from cells of the artery wall.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/genética , Antirreumáticos/farmacología , Colestanotriol 26-Monooxigenasa/genética , Células Espumosas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Metotrexato/farmacología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina/metabolismo , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/prevención & control , Células Cultivadas , Colestanotriol 26-Monooxigenasa/metabolismo , Colesterol/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Interacciones Farmacológicas , Células Espumosas/citología , Células Espumosas/metabolismo , Humanos , Interferón gamma/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , ARN Mensajero/metabolismo , Receptor de Adenosina A2A/metabolismo

RESUMEN

It is generally believed that vitamin E is absorbed along with chylomicrons. However, we previously reported that human colon carcinoma Caco-2 cells use dual pathways, apolipoprotein B (apoB)-lipoproteins and HDLs, to transport vitamin E. Here, we used primary enterocytes and rodents to identify in vivo vitamin E absorption pathways. Uptake of [(3)H]alpha-tocopherol by primary rat and mouse enterocytes increased with time and reached a maximum at 1 h. In the absence of exogenous lipid supply, these cells secreted vitamin E with HDL. Lipids induced the secretion of vitamin E with intermediate density lipoproteins, and enterocytes supplemented with lipids and oleic acid secreted vitamin E with chylomicrons. The secretion of vitamin E with HDL was not affected by lipid supply but was enhanced when incubated with HDL. Microsomal triglyceride transfer protein inhibition reduced vitamin E secretion with chylomicrons without affecting its secretion with HDL. Enterocytes from Mttp-deficient mice also secreted less vitamin E with chylomicrons. In vivo absorption of [(3)H]alpha-tocopherol by mice after poloxamer 407 injection to inhibit lipoprotein lipase revealed that vitamin E was associated with triglyceride-rich lipoproteins and small HDLs containing apoB-48 and apoA-I. These studies indicate that enterocytes use two pathways for vitamin E absorption. Absorption with chylomicrons is the major pathway of vitamin E absorption. The HDL pathway may be important when chylomicron assembly is defective and can be exploited to deliver vitamin E without increasing fat consumption.

Asunto(s)

Enterocitos/metabolismo , Absorción Intestinal/fisiología , Vitamina E/metabolismo , Animales , Ácidos y Sales Biliares/fisiología , Proteínas Portadoras/antagonistas & inhibidores , Lipoproteínas HDL/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Micelas , Ácido Oléico/fisiología , Ratas , Ratas Sprague-Dawley

RESUMEN

Pluronic L81 (PL81) inhibits fat absorption, and other Pluronic copolymers help overcome drug resistance in cancer cells. To understand how PL81 acts, we synthesized a radiolabeled analog, [14C]PL81, and showed that it was structurally similar to PL81 based on (1)H NMR as well as mass spectrometric analysis. [14C]PL81 inhibited the secretion of chylomicrons (CMs), lipoproteins essential for fat absorption, by differentiated Caco-2 cells similar to PL81. Moreover, PL81 competed with the cellular uptake of [14C]PL81. Thus, [14C]PL81 and PL81 behave similarly in these physiologic assays. Uptake of [14C]PL81 by Caco-2 cells was concentration-, time-, and temperature-dependent and occurred mainly from the apical side. Intracellularly, it was assimilated in the cytosol. Cells excreted PL81 toward the apical side via a pathway partially sensitive to verapamil. Small amounts were secreted toward the basolateral side unassociated with CM, and this secretion was unaffected by the inhibition of CM assembly. Nonetheless, PL81 significantly inhibited the secretion of triacylglycerols (TGs) and phospholipids as part of CM. PL81-treated cells showed decreased activity of microsomal triglyceride transfer protein and accumulated more TGs, but not phospholipids, in their cytosol. We propose that Pluronic copolymers act by interfering with the export of molecules from the cytosol. They inhibit fat absorption by decreasing TG transport to the endoplasmic reticulum and increase drug efficacy against cancer cells by competing for their excretion.

Asunto(s)

Quilomicrones/metabolismo , Citosol/metabolismo , Poloxámero/farmacología , Tensoactivos/farmacología , Triglicéridos/metabolismo , Transporte Biológico , Células CACO-2 , Centrifugación por Gradiente de Densidad , Retículo Endoplásmico/metabolismo , Humanos , Modelos Químicos , Fracciones Subcelulares , Factores de Tiempo

RESUMEN

In hepatocytes, vitamin E is secreted via the efflux pathway and is believed to associate with apolipoprotein B (apoB)-lipoproteins extracellularly. The molecular mechanisms involved in the uptake, intracellular trafficking, and secretion of dietary vitamin E by the intestinal cells are unknown. We observed that low concentrations of Tween-40 were better for the solubilization and delivery of vitamin E to differentiated Caco-2 cells, whereas high concentrations of Tween-40 and sera inhibited this uptake. Vitamin E uptake was initially rapid and then reached saturation. Subcellular localization revealed that vitamin E primarily accumulated in microsomal membranes. Oleic acid (OA) treatment, which induces chylomicron assembly and secretion, decreased microsomal membrane-bound vitamin E in a time-dependent manner. To study secretion, differentiated Caco-2 cells were pulse-labeled with vitamin E and chased in the presence and absence of OA. In the absence of OA, vitamin E was associated with intestinal high density lipoprotein (I-HDL), whereas OA-treated cells secreted vitamin E with I-HDL and chylomicrons. No extracellular transfer of vitamin E between these lipoproteins was observed. Glyburide, an antagonist of ABCA1, partially inhibited its secretion with I-HDL, whereas plasma HDL increased vitamin E efflux. An antagonist of microsomal triglyceride transfer protein, brefeldin A, and monensin specifically inhibited vitamin E secretion with chylomicrons. These studies indicate that vitamin E taken up by Caco-2 cells is stored in the microsomal membranes and secreted with chylomicrons and I-HDL. Transport via I-HDL might contribute to vitamin E absorption in patients with abetalipoproteinemia receiving large oral doses of the vitamin.

Asunto(s)

Diferenciación Celular , Vitamina E/metabolismo , Apolipoproteínas A/metabolismo , Transporte Biológico , Células CACO-2 , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Ésteres del Colesterol/metabolismo , Quilomicrones/metabolismo , Quilomicrones/fisiología , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citología , Lipoproteínas HDL/metabolismo , Modelos Biológicos , Transducción de Señal/fisiología , Vitamina E/farmacocinética

RESUMEN

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K(m) values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9+/-2.1 microM and 13.9+/-0.3 microM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits approximately 85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl-oleate formation without influencing the retinyl-palmitate formation. Using this inhibitor, we estimate that approximately 64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.

Asunto(s)

Diacilglicerol O-Acetiltransferasa/metabolismo , Retinol O-Graso-Aciltransferasa/metabolismo , Vitamina A/metabolismo , Aciltransferasas/metabolismo , Animales , Células CACO-2 , Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Compuestos Heterocíclicos con 1 Anillo/farmacología , Humanos , Cinética

RESUMEN

The present study provides a new understanding about the mechanisms involved in cholesterol absorption by the intestinal cells. Contrary to general belief, our data show that newly absorbed cholesterol is neither immediately available for secretion with apoB lipoproteins nor exclusively secreted as part of chylomicrons. Based on our data, cholesterol transport by enterocytes can be broadly classified into two independently modulated, apoB-dependent and -independent, pathways. Cholesterol secretion by the apoB-dependent pathway is induced by oleic acid, is repressed by microsomal triglyceride transfer protein inhibitors, and occurs only with larger apoB-containing lipoproteins. ApoB-independent pathways do not require microsomal triglyceride transfer protein and involve efflux mediated by ABCA1, high density lipoprotein assembly, and possibly other unknown mechanisms. There are at least two different metabolic pools of cholesterol. The newly absorbed and pre-absorbed cholesterol are preferentially secreted via apoB-independent and apoB-dependent pathways, respectively. In contrast to compartmentalization for secretion, these two metabolic pools are equally accessible for cellular esterification. The esterified cholesterol is mainly secreted by the apoB-dependent pathway, whereas both the pathways are involved in the secretion of free cholesterol. Thus, enterocytes transport exogenous cholesterol by several independently regulated pathways raising the possibility that targeting of apoB-independent pathways may result in selective inhibition of cholesterol transport without affecting triglyceride transport.

Asunto(s)

Colesterol/metabolismo , Mucosa Intestinal/metabolismo , Animales , Transporte Biológico , Femenino , Humanos , Lipoproteínas/metabolismo , Ratas , Células Tumorales Cultivadas

RESUMEN

Microsomal triglyceride transfer protein (MTP) is a heterodimeric protein that transfers neutral lipids between membranes in vitro. Absence of this lipid transfer activity in the microsomes of abetalipoproteinemia patients established its pivotal function in lipoprotein assembly. Recent studies indicate that the lipid transfer activity is involved in importing triglycerides into the lumen of the endoplasmic reticulum. In addition to its lipid transfer activity, MTP physically interacts with apoB. This led to speculation that MTP may act as a chaperone. It remains to be determined whether the binding of MTP to apoB plays a role in either proper folding or net lipidation of nascent apoB. Both functions, lipid transfer and apoB binding, may be involved in the initial step of lipidation of nascent apoB resulting in the synthesis of primordial lipoprotein particles. Furthermore, it has been shown that MTP stably associates with lipid vesicles. The lipid-associated MTP may be important in core expansion of primordial lipoproteins. In summary, three independent functions (lipid transfer, apoB binding and membrane association) of MTP have been identified. Here, we propose these functions are carried out by a combination of different structural motifs. Based on sequence homology with lipovitellin, the M subunit of MTP is predicted to contain three beta-sheets (A, C, and N) and one alpha-helical domain. The A- and C-sheets may be involved in lipid transfer, the N-sheet and the helical domain in apoB binding, and the N- and A-sheets in membrane association. It is also speculated that MTP may function in physiologic processes beyond lipoprotein assembly.

Asunto(s)

Proteínas Portadoras/fisiología , Animales , Proteínas Portadoras/química , Humanos , Conformación Proteica