RESUMEN
Cellular uptake of vitamin A, production of visual chromophore and triglyceride homeostasis in adipocytes depend on two representatives of the vertebrate N1pC/P60 protein family, lecithin:retinol acyltransferase (LRAT) and HRAS-like tumor suppressor 3 (HRASLS3). Both proteins function as lipid-metabolizing enzymes but differ in their substrate preferences and dominant catalytic activity. The mechanism of this catalytic diversity is not understood. Here, by using a gain-of-function approach, we identified a specific sequence responsible for the substrate specificity of N1pC/P60 proteins. A 2.2-Å crystal structure of the HRASLS3-LRAT chimeric enzyme in a thioester catalytic intermediate state revealed a major structural rearrangement accompanied by three-dimensional domain swapping dimerization not observed in native HRASLS proteins. Structural changes affecting the active site environment contributed to slower hydrolysis of the catalytic intermediate, supporting efficient acyl transfer. These findings reveal structural adaptation that facilitates selective catalysis and mechanism responsible for diverse substrate specificity within the LRAT-like enzyme family.
Asunto(s)
Aciltransferasas/metabolismo , Fosfolipasas A2 Calcio-Independiente/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Vitamina A/metabolismo , Animales , Humanos , Ratones , Ratones Transgénicos , Modelos Moleculares , Conformación Proteica , Retinol O-Graso-Aciltransferasa/metabolismo , Especificidad por SustratoRESUMEN
Absorption of a photon by a rhodopsin or cone-opsin pigment isomerizes its 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which dissociates after a brief period of activation. Light sensitivity is restored to the resulting apo-opsin when it recombines with another 11-cis-RAL. Conversion of all-trans-RAL to 11-cis-RAL is carried out by an enzyme pathway called the visual cycle in cells of the retinal pigment epithelium. A second visual cycle is present in Müller cells of the retina. The retinol isomerase for this noncanonical pathway is dihydroceramide desaturase (DES1), which catalyzes equilibrium isomerization of retinol. Because 11-cis-retinol (11-cis-ROL) constitutes only a small fraction of total retinols in an equilibrium mixture, a subsequent step involving selective removal of 11-cis-ROL is required to drive synthesis of 11-cis-retinoids for production of visual chromophore. Selective esterification of 11-cis-ROL is one possibility. Crude homogenates of chicken retinas rapidly convert all-trans-ROL to 11-cis-retinyl esters (11-cis-REs) with minimal formation of other retinyl-ester isomers. This enzymatic activity implies the existence of an 11-cis-specific retinyl-ester synthase in Müller cells. Here, we evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this 11-cis-RE-synthase. MFAT exhibited much higher catalytic efficiency as a synthase of 11-cis-REs versus other retinyl-ester isomers. Further, we show that MFAT is expressed in Müller cells. Finally, homogenates of cells coexpressing DES1 and MFAT catalyzed the conversion of all-trans-ROL to 11-cis-RP, similar to what we observed with chicken-retina homogenates. MFAT is therefore an excellent candidate for the retinyl-ester synthase that cooperates with DES1 to drive synthesis of 11-cis-retinoids by mass action.
Asunto(s)
Acetiltransferasas/metabolismo , Células Ependimogliales/enzimología , Enzimas Multifuncionales/metabolismo , Retinol O-Graso-Aciltransferasa/metabolismo , Animales , Catálisis , Bovinos , Pollos , Opsinas de los Conos/metabolismo , Ésteres/química , Ácidos Grasos/química , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Células HEK293 , Humanos , Cinética , Ratones , Opsinas/metabolismo , Retina/metabolismoRESUMEN
Measuring concentrations of the differentiation-promoting hormone retinoic acid (RA) in glioblastoma tissues would help to understand the reason why RA treatment has been inefficient in clinical trials involving brain tumor patients. Here, we apply a recently established extraction and measurement protocol to screen glioblastoma tissues for the levels of the RA precursor retinol and biologically active RA. Combining this approach with mRNA analyses of 26 tumors and 8 normal brains, we identify a multifaceted disturbance of RA synthesis in glioblastoma, involving multiple aldehyde dehydrogenase 1 family and retinol dehydrogenase enzymes. Through database studies and methylation analyses, we narrow down chromosomal deletions and aberrant promoter hypermethylation as potential mechanisms accounting for these alterations. Employing chromatin immunoprecipitation analyses and cell-culture studies, we further show that chromatin at RA target genes is poised to RA substitution, but most glioblastoma cell cultures are completely resistant to RA treatment. This paradoxical RA response is unrelated to alternative RA signaling through the fatty acid-binding protein 5/peroxisome proliferator-activated receptor delta axis. Our data suggest a multifaceted disturbance of RA synthesis in glioblastoma and contribute to reconsider current RA treatment strategies.
Asunto(s)
Neoplasias Encefálicas/complicaciones , Encéfalo/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Glioblastoma/complicaciones , Tretinoina/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Encéfalo/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Metilación de ADN , Bases de Datos Bibliográficas/estadística & datos numéricos , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/metabolismo , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Retinal-Deshidrogenasa/metabolismo , Retinoides/farmacología , Retinol O-Graso-Aciltransferasa/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Approximately 80-90% of all retinoids in the body are stored as retinyl esters (REs) in the liver. Adipose tissue also contributes significantly to RE storage. The present studies, employing genetic and nutritional interventions, explored factors that are responsible for regulating RE accumulation in the liver and adipose tissue and how these influence levels of retinoic acid (RA) and RA-responsive gene expression. Our data establish that acyl-CoA:retinol acyltransferase (ARAT) activity is not involved in RE synthesis in the liver, even when mice are nutritionally stressed by feeding a 25-fold excess retinol diet or upon ablation of cellular retinol-binding protein type I (CRBPI), which is proposed to limit retinol availability to ARATs. Unlike the liver, where lecithin:retinol acyltransferase (LRAT) is responsible for all RE synthesis, this is not true for adipose tissue where Lrat-deficient mice display significantly elevated RE concentrations. However, when CrbpI is also absent, RE levels resemble wild-type levels, suggesting a role for CrbpI in RE accumulation in adipose tissue. Although expression of several RA-responsive genes is elevated in Lrat-deficient liver, employing a sensitive liquid chromatography tandem mass spectrometry protocol and contrary to what has been assumed for many years, we did not detect elevated concentrations of all-trans-RA. The elevated RA-responsive gene expression was associated with elevated hepatic triglyceride levels and decreased expression of Pparδ and its downstream Pdk4 target, suggesting a role for RA in these processes in vivo.
Asunto(s)
Tejido Adiposo Blanco/metabolismo , Hígado/metabolismo , Retinoides/metabolismo , Animales , Epidídimo/metabolismo , Esterificación , Ésteres , Femenino , Expresión Génica , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , PPAR delta/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Retinol O-Graso-Aciltransferasa/genética , Retinol O-Graso-Aciltransferasa/metabolismo , Proteínas Celulares de Unión al Retinol/genética , Proteínas Celulares de Unión al Retinol/metabolismo , Triglicéridos/metabolismoRESUMEN
GS2 (PNPLA4; iPLAeta) is the smallest member of the patatin-like family of phospholipases (PNPLA). It was initially identified by its ability to hydrolyze retinylesters (RE) in cell homogenates, and was later found to esterify retinol using a variety of acyl donors. In the present study we set out to determine its cellular function and examined its impact on RE status in 293T cells transfected with GS2, GS2-M1 (a non-translatable mutant of GS2) and empty vector, in fibroblasts isolated from normal and GS2-null donors and in SCC12b and in a somatic cell knock-out of GS2 (SCC12b-GS2(neo/-)), that we generated by homologous recombination. At 50nM medium retinol, GS2 had no significant impact on RE accumulation. However, at 2muM retinol, GS2 promoted a 1.6- to 5-fold increase in RE accumulation. To verify role of GS2 as a catalyst, RE levels were measured in 293T transfected wild type GS2, catalytic dyad mutants devoid of enzymatic activity, or alanine substitution mutants spanning the entire GS2 sequence. Surprisingly, every GS2 mutant promoted RE accumulation. This activity was also observed in the GS2 paralogues and rat orthologue. The data demonstrate that within the context of the cell GS2 promotes RE accumulation and may do so either as a catalyst or as a regulatory protein that enhances RE formation catalyzed by other acyl transferases.
Asunto(s)
Biocatálisis , Ésteres/metabolismo , Proteínas/metabolismo , Retinol O-Graso-Aciltransferasa/metabolismo , Tretinoina/análogos & derivados , Aciltransferasas/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular , Esterificación , Exones/genética , Fibroblastos/enzimología , Fibroblastos/patología , Regulación Enzimológica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Lipasa , Ratones , Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Recombinación Genética/genética , Eliminación de Secuencia , Tretinoina/metabolismoRESUMEN
The mechanism of the intestinal esterification of retinol has been obscure. Recently, an acyl-Coenzyme A (CoA):retinol acyltransferase (ARAT) was found in rat intestinal microsomes, and experiments were therefore conducted to determine whether a corresponding enzyme exists in human small intestine. When microsomes were incubated with [3H]retinol and palmitoyl-CoA, or retinol and [1-14C]palmitoyl-CoA, radioactive retinyl palmitate was formed as identified by alumina column chromatography and reverse-phase high-pressure liquid chromatography. Heating the microsomes for 30 min at 60 degrees C resulted in loss of activity. The esterification was negligible without exogenous acyl-CoA and markedly stimulated by palmitoyl-, oleoyl-, and stearoyl-CoA in concentrations up to 20 microM. The acyl-CoA was successfully replaced by an acyl-CoA generating system, but not by unactivated palmitate (2.5-200 microM). The assay was dependent on the presence of albumin with optimum activity at 2-10 mg/ml. The optimal retinol concentration was 20-30 microM and pH approximately 7.4. The esterifying activity was completely inhibited by 8 mM of taurocholate and to 90% by 1 mM of 5,5'-dithiobis(2-nitrobenzoic acid). Activity was found throughout the small intestine. In jejunum the rate of retinol esterification was: 3.44 +/- 2.24 nmol [3H]retinyl ester formed . mg microsomal protein-1 . min-1 (mean +/- SD, n = 12). The corresponding activity in whole homogenates of biopsies were 1.17 +/- 0.28 (n = 8). It is concluded that human small intestine contains a microsomal acyl-CoA:retinol acyltransferase. Due to its high activity in vitro this enzyme is likely to be responsible for the intestinal esterification of retinol.
Asunto(s)
Aciltransferasas/fisiología , Mucosa Intestinal/enzimología , Microsomas/enzimología , Vitamina A/metabolismo , Aciltransferasas/antagonistas & inhibidores , Ácido Ditionitrobenzoico/farmacología , Esterificación , Ácidos Grasos/metabolismo , Humanos , Técnicas In Vitro , Retinol O-Graso-Aciltransferasa , Ácido Taurocólico/farmacologíaAsunto(s)
Neuroglía/metabolismo , Retinaldehído/síntesis química , Retinol O-Graso-Aciltransferasa/metabolismo , Proteínas Celulares de Unión al Retinol/síntesis química , Vitamina A/biosíntesis , Animales , Bovinos , Células Cultivadas , Pollos , Cromatografía Líquida de Alta Presión , Ésteres/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , RatasRESUMEN
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 , CinéticaRESUMEN
This study was conducted to examine the in vivo uptake and metabolism of natural retinoids by N-methyl-N-nitrosourea-induced mammary carcinomas. In this study, endogenous retinol and retinyl esters were present in normal mammary epithelial cells, but were undetectable in N-methyl-N-nitrosourea-induced mammary carcinomas in rats as determined by high-pressure liquid chromatography. No differences were found in plasma levels of retinol, in liver retinyl esters, or total content of vitamin A between tumor-bearing and control animals. Administered labeled retinol was taken up and esterified by normal mammary epithelial cells. Tumor-bearing rats were given injections i.p. of either [3H]retinol or [3H]retinoic acid. Radioactivity increased progressively with time in liver and other tissues except in breast tumor, where the uptake fluctuated over the 8 days after the injection of [3H]retinol; in mammary tumors practically no metabolism of [3H]retinol occurred, while in other tissues extensive esterification was detectable. In contrast, in animals given injections of [3H]retinoic acid, the uptake and metabolism of the label in the breast tumors paralleled with those found in other tissues. Neither the activity of acyl coenzyme A:retinol acyl transferase nor the activity of retinyl ester hydrolase was altered in the mammary tumor compared to the normal mammary gland. On the other hand, a significant decrease in the retinal oxidase activity was found in tumor tissue compared to normal mammary tissue. Since no esterification of [3H]retinol occurred in vivo despite the presence of acyl coenzyme A:retinol acyl transferase activity, it is possible that a specific defect in the cellular uptake of retinol may exist in N-methyl-N-nitrosourea-induced mammary carcinomas.
Asunto(s)
Neoplasias Mamarias Experimentales/metabolismo , Tretinoina/metabolismo , Vitamina A/metabolismo , Aciltransferasas/análisis , Animales , Hidrolasas de Éster Carboxílico/análisis , Femenino , Hígado/análisis , Glándulas Mamarias Animales/análisis , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/inducido químicamente , Metilnitrosourea , Ratas , Ratas Endogámicas , Retinol O-Graso-Aciltransferasa , Vitamina A/análisisRESUMEN
The present study was conducted to examine esterification of retinol by testicular microsomes. The microsomes were isolated from rat testes and were incubated under varying assay conditions with [3H]retinol. [3H]Retinylpalmitate was identified by reversed-phase high-performance liquid chromatography as an esterified product. The rate of esterification was increased by the addition of a fatty acyl-CoA. Coenzyme A esters of oleic, palmitic and stearic acids were equally effective substrates for retinol esterification. A 17-fold increase was observed in the presence of palmitoyl-CoA when microsomes had been pretreated with hydroxylamine, a reagent that reacts with coenzyme A thioesters to form hydroxamic acids. The esterifying activity was stimulated by the addition of dithiothreitol (4 mM) and fatty acid-free bovine serum albumin (1 mg/ml). The optimal concentrations for retinol and palmitoyl-CoA were 40 microM and 30-40 microM, respectively. The enzyme activity was inhibited by p-hydroxymercuribenzoate, sodium taurocholate and 5,5'-dithiobis-(2-nitrobenzoic acid), but not by EDTA. The enzyme activity was highest in microsomes (36%). However, some activity was present in mitochondria (29%). These results clearly show the presence of a fatty acyl-CoA: retinol acyltransferase that catalyzes the esterification of retinol in rat testes.
Asunto(s)
Aciltransferasas/metabolismo , Testículo/enzimología , Aciltransferasas/aislamiento & purificación , Animales , Hidroxilamina , Hidroxilaminas/farmacología , Cinética , Masculino , Microsomas/enzimología , Ratas , Ratas Endogámicas , Retinol O-Graso-Aciltransferasa , Fracciones Subcelulares/enzimologíaRESUMEN
Chronic dietary administration of 3,3',4,4',5,5'-hexabromobiphenyl (HBB), 1 mg/kg diet, caused a decrease in retinol (20-fold) and retinyl esters (23-fold) in the livers of female rats, but resulted in a 6.4-fold increase in retinol and 7.4-fold increase in retinyl esters in the kidneys. Liver acyl-CoA:retinol acyltransferase and retinyl palmitate hydrolase activities were reduced while serum concentration of retinol was unaffected by HBB feeding. Metabolism of a physiological dose of [11-3H]retinyl acetate (10 micrograms), was examined in rats fed either vitamin A-adequate diet, or marginal amounts of vitamin A, or vitamin A-adequate diet containing HBB. A 13-fold greater amount of the administered vitamin A was found in kidneys of HBB-treated rats. In rats fed adequate or low amounts of vitamin A, kidney radioactivity was primarily in the retinol fraction, while in HBB-fed rats the radioactivity was associated mostly with retinyl esters. Fecal and urinary excretion of radioactivity was greatly increased in HBB-treated rats. Chronic HBB feeding results in a loss of ability of liver to store vitamin A, and severely alters the uptake and metabolism of vitamin A in the kidneys. We conclude that HBB causes major disturbances in the regulation of vitamin A metabolism.
Asunto(s)
Bifenilos Polibrominados/metabolismo , Vitamina A/metabolismo , Aciltransferasas/metabolismo , Animales , Hidrolasas de Éster Carboxílico/metabolismo , Diterpenos , Femenino , Riñón/metabolismo , Hígado/metabolismo , Bifenilos Polibrominados/farmacología , Ratas , Retinol O-Graso-Aciltransferasa , Ésteres de Retinilo , Vitamina A/análogos & derivadosRESUMEN
Cholesterol and retinol are both esterified with long-chain fatty acid within the mucosal cells of the small intestine. The reactions are catalyzed by microsomal acyl-CoA:cholesterol and acyl-CoA:retinol acyltransferases (EC 2.3.1.26, and EC 2.3.1.-, respectively). To gain more insight into the physiological importance of these acyltransferases, they were studied in villous and crypt cells from rats either fasting or on diets which varied in fat and cholesterol content. Both enzymes had a higher activity in villous than in crypt cells. The activities in villous cells varied with feeding and fasting and the composition of diet when the animals were killed postprandially. Acyl-CoA:cholesterol acyltransferase activity went up upon cholesterol feeding whereas retinol acyltransferase in the mucosa was reduced by high-fat diets. The liver cholesterol acyltransferase activity varied with diet, it increased with both cholesterol and fat feeding, whereas retinol acyltransferase activity remained relatively constant. The results obtained suggest that different diets are of importance for cholesterol and retinol acyltransferase activities both in the intestinal mucosa and in the liver. The variation in activities of the two acyltransferases suggests that they may be different enzymes.
Asunto(s)
Aciltransferasas/metabolismo , Dieta , Mucosa Intestinal/enzimología , Intestino Delgado/enzimología , Hígado/enzimología , Esterol O-Aciltransferasa/metabolismo , Aciltransferasas/fisiología , Animales , Colesterol en la Dieta/farmacología , Ayuno , Absorción Intestinal , Masculino , Ratas , Ratas Endogámicas , Retinol O-Graso-Aciltransferasa , Esterol O-Aciltransferasa/fisiología , Vitamina A/farmacologíaRESUMEN
Since the factors regulating retinol esterification by acyl-CoA:retinol acyltransferase are poorly understood, we studied the age-related variations in acyl-CoA:retinol acyltransferase activity in hairless mice. Epidermis and liver were collected at intervals from birth to adolescence (0-6 weeks). Vitamin A was analyzed by high-performance liquid chromatography and acyl-CoA:retinol acyltransferase by an in vitro radioincubation assay of microsomes. Epidermal vitamin A (retinol plus retinyl esters) increased 8-10 times after birth and by the age of 3 weeks adult values were attained. This increase was accompanied by a 2-fold increase in acyl-CoA:retinol acyltransferase activity in the epidermis between 3 days and 6 weeks of age. In young animals the dependence of acyl-CoA:retinol acyltransferase on exogenous co-substrate (palmitoyl-CoA) was also lower than in adult animals. Although a pronounced age-related accumulation of retinol was recorded in the liver, the activity of acyl-CoA:retinol acyltransferase did not increase with age and there was no change in the dependence of acyl-CoA:retinol acyltransferase on exogenous palmitoyl-CoA.
Asunto(s)
Aciltransferasas/metabolismo , Envejecimiento/metabolismo , Epidermis/metabolismo , Hígado/metabolismo , Vitamina A/metabolismo , Animales , Epidermis/efectos de los fármacos , Hígado/efectos de los fármacos , Ratones , Ratones Pelados , Microsomas/enzimología , Microsomas Hepáticos/enzimología , Palmitoil Coenzima A/farmacología , Retinol O-Graso-AciltransferasaRESUMEN
In an attempt to characterize the enzyme(s) responsible for retinol esterification in hairless mouse epidermis, various subcellular fractions were incubated with [3H]retinol and the reaction products (retinyl esters) isolated by high-performance liquid chromatography. The microsomal fraction exhibited the highest esterifying activity and was stimulated by the addition of palmitoyl-CoA and dithiothreitol, but not by palmitic acid. Saturation kinetics with an apparent Km of about 6 microM for retinol were noted. Experiments with competitive and noncompetitive inhibitors of [3H]retinol esterification established that the epidermal enzyme was an acyl-CoA:retinol acyltransferase (ARAT; EC 2.3.1.76). The specificity for retinol was not absolute; a few closely related vitamin A alcohols were equally good substrates. The ARAT activity was not significantly altered by physiologic variations in the epidermal vitamin A content. In conclusion, mouse epidermis expresses ARAT activity which may be of importance for the regulation of vitamin A metabolism at the cellular level.
Asunto(s)
Epidermis/ultraestructura , Vitamina A/metabolismo , Aciltransferasas/metabolismo , Animales , Unión Competitiva , Diterpenos , Ésteres/metabolismo , Ratones , Microsomas/metabolismo , Retinol O-Graso-Aciltransferasa , Ésteres de Retinilo , Vitamina A/análogos & derivados , Vitamina A/biosíntesisRESUMEN
Keratinocytes from three different layers of epidermis (stratum basale, stratum spinosum, and stratum granulosum/corneum) were shown by high-performance liquid chromatography to contain retinol, 3,4-didehydroretinol and several fatty acyl esters thereof. The concentration of unesterified congeners increased 1.8-2.8 times from the inner to the outer layers of epidermis, while the corresponding increase in fatty acyl esters was 4.0-6.5 times. Together the esters represented 71% of the total vitamin A content in stratum granulosum/corneum as compared to 54% in stratum basale. The in situ synthesis of fatty acyl esters of retinol and 3,4-didehydroretinol (vitamin A2) was studied by addition of [3H]retinol to organ-cultured human breast skin. The radioactive compounds appearing in the epidermis after 48 h were, in order of abundance, retinyl esters, retinol, 3,4-didehydroretinyl esters, and 3,4-didehydroretinol. Studies at the subcellular level demonstrated the highest esterifying activity in the microsomal fraction. The enzyme catalyzing the reaction, acyl CoA:retinol acyltransferase (ARAT; EC 2.3.1.76), had a pH optimum of 5.5-6.0, which differs from that of ARAT in other tissues. ARAT activities in microsomes from different layers of epidermis were similar, but, owing to a presumed pH gradient in upper epidermis, the in vivo esterification of vitamin A may be enhanced in terminally differentiating keratinocytes. The mean ARAT activities in basal cell carcinomas and squamous cell carcinomas were less than 50% of the control values, and the relative amounts of retinyl esters were significantly lower than normal. We suggest that the esterification of vitamin A may also be of importance in relation to pathologic keratinocyte differentiation.
Asunto(s)
Epidermis/metabolismo , Queratinocitos/citología , Vitamina A/metabolismo , Aciltransferasas/metabolismo , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Separación Celular , Células Epidérmicas , Esterificación , Humanos , Masculino , Microsomas/metabolismo , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Concentración Osmolar , Valores de Referencia , Retinol O-Graso-Aciltransferasa , Enfermedades de la Piel/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
Vitamin A is stored in cells as long-chain fatty acyl esters of retinol. Esterification in many tissues is catalyzed in part by acyl-CoA:retinol acyltransferase (ARAT). Since the lacrimal gland contains stores of retinyl esters, it was the goal of this study to determine whether the lacrimal gland contains ARAT activity. Rabbit lacrimal gland microsomes incubated with 3H-retinol synthesized retinyl esters. The reaction rate was stimulated 30-fold in the presence of a fatty acyl-CoA generating system, producing a mixture of esters including retinyl laurate, retinyl linoleate, retinyl palmitate, and retinyl stearate as determined by reverse-phase HPLC. Retinyl palmitate was synthesized at 1944 pmole/mg protein/30 min, representing 50% of total ester synthesis, and this activity was directly proportional to microsomal protein concentration. In the presence of 180 microM 3H-retinol and 100 microM palmitoyl-CoA, retinyl palmitate was synthesized at 175-220 pmole/mg/min, and the reaction fit Michaelis-Menten kinetics as a function of retinal concentration (theoretical Vmax = 329.4 pmole/mg/min). Lauroyl CoA and stearoyl CoA, but not linoleoyl CoA, were as effective as palmitoyl CoA as substrates for the reaction. The enzyme activity was inhibited by p-chloromercuriphenyl sulfonic acid and Na-taurocholate. The data show that the lacrimal gland synthesizes retinyl esters and that the characteristics of synthesis are consistent with the presence of acyl-CoA:retinol acyltransferase in lacrimal gland.
Asunto(s)
Aciltransferasas/metabolismo , Aparato Lagrimal/metabolismo , Vitamina A/metabolismo , Acilcoenzima A/farmacología , Aciltransferasas/antagonistas & inhibidores , Animales , Cromatografía Líquida de Alta Presión , Diterpenos , Esterificación , Cinética , Aparato Lagrimal/enzimología , Microsomas/enzimología , Microsomas/metabolismo , Palmitoil Coenzima A/farmacología , Conejos , Retinol O-Graso-Aciltransferasa , Ésteres de Retinilo , Vitamina A/análogos & derivados , Vitamina A/biosíntesisRESUMEN
UVB irradiation depletes all-trans-retinol (ROL) and all-trans-retinyl esters (RE) from the hairless mouse epidermis. Prevention of this may be of relevance in counter-acting the long-term side effects of UVB exposure. We studied the effects of a topical treatment with natural retinoids before and after UVB exposure on three parameters involved in vitamin A metabolism: the amount of epidermal ROL and RE, the level of functional cellular retinol-binding protein I (CRBP-I), which is likely to protect ROL from UVB, as well as the cytosolic and microsomal enzyme activities which generate ROL and RE, i.e. all-trans-retinaldehyde (RAL) reductase, acylCoA:retinol acyltransferase (ARAT) and retinyl-ester hydrolase (REH). Topical pretreatment with retinoids promoted a dramatic increase of epidermal ROL, RE and CRBP-I levels, a transient increase of RAL reductase and ARAT activities as well as a decreased activity of REH, indicating a direction of epidermal vitamin A metabolism toward storage. In untreated mice UVB irradiation induced a depletion of epidermal ROL and RE in 10 min and a 50% decrease of CRBP-I after 24 h. In mice treated with topical retinoids, and then exposed to UVB, epidermal RE levels were higher than in vehicle-treated, nonirradiated mice. In contrast, ROL was as much depleted after UVB in pretreated as in untreated animals in spite of an induction of CRBP-I, indicating that CRBP-I does not actually protect ROL from UVB-induced depletion in this model. However, the reconstitution of both epidermal ROL and RE, after their depletion induced by UVB, was accelerated by previous topical treatment with RAL. Our results indicate that topical delivery of retinoids partly counteracts UVB-induced vitamin A depletion and promotes recovery.
Asunto(s)
Epidermis/enzimología , Epidermis/efectos de la radiación , Retinoides/metabolismo , Rayos Ultravioleta , Vitamina A/metabolismo , Aciltransferasas/metabolismo , Animales , Hidrolasas de Éster Carboxílico/metabolismo , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Epidermis/metabolismo , Ésteres , Femenino , Ratones , Ratones Endogámicos , Ratones Desnudos , Retinoides/administración & dosificación , Retinol O-Graso-Aciltransferasa , Proteínas de Unión al Retinol/metabolismo , Proteínas Celulares de Unión al Retinol , Vitamina A/análisisRESUMEN
The conversion of beta-carotene to retinal and the succeeding metabolic process of the retinal leading to production of retinol and retinyl esters are the prerequisite for the utilization of beta-carotene as a provitamin A. These processes are participated by beta-carotene cleavage enzyme, retinal reductase and retinol esterifying enzyme(s) in the small intestine. To examine whether these enzymes exhibit the coordinated distribution in the villus, we have used the cryostat sectioning technique to quantify the activities of beta-carotene cleavage enzyme, retinal reductase and retinol esterifying enzymes along the villus-crypt axis in 8-day-old chick duodenum. The beta-carotene cleavage enzyme activity was very low in the crypt and gradually increased, reaching a maximum in the mid-villus. The villus-crypt gradient of the beta-carotene cleavage enzyme activity corresponded with those of retinal reductase activity and lecithin: retinol acyltransferase (LRAT) activity, but distinct from that of acyl-CoA: retinol acyltransferase (ARAT) activity. Furthermore, the distribution of the content of retinyl esters was similar to that of LRAT activity. These results suggest that the beta-carotene cleavage enzyme is coordinately distributed along the villus-crypt axis with retinal reductase and LRAT, the two enzymes which require cellular retinol-binding protein, typeII (CRBPII) as the donor of the substrate.
Asunto(s)
Aciltransferasas/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Duodeno/enzimología , Vitamina A/metabolismo , beta Caroteno/metabolismo , Animales , Anticarcinógenos/análisis , Pollos , Diterpenos , Duodeno/química , Retinol O-Graso-Aciltransferasa , Ésteres de Retinilo , Vitamina A/análogos & derivados , Vitamina A/análisisRESUMEN
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters the turnover of vitamin A in the body and inhibits the normal hepatic accumulation of dietary vitamin A. Vitamin A is absorbed in the small intestine, where it is incorporated into chylomicrons as retinyl esters for release into the lymph and further distributed via blood to the liver for storage. The aim of the present study was to investigate if the decreased hepatic vitamin A levels in TCDD-exposed rats could be due to impaired intestinal absorption of vitamin A via lymph. Male Sprague-Dawley rats were given a single oral dose of TCDD (10 microg/kg). Five days after administration, the main intestinal lymph duct of the rats was cannulated. After a 24-h recovery from surgery, the rats were each given a single dose of [3H]retinol in corn oil via gavage and the lymph was collected for 24 h. The cumulative radiolabel recovered in the intestinal lymph was significantly lower in TCDD-treated than in control rats during the first 6 h of absorption. However, no significant differences in radiolabel recovered in lymph were seen when looking at the entire 24-h collection period. In the intestinal mucosa, retinol esterification catalyzed by the enzyme lecithin:retinol acyl transferase (LRAT) or acyl coenzyme A (CoA):retinol transferase (ARAT) was not statistically different between the groups. However, mucosal retinyl palmitate levels were significantly increased in TCDD-treated rats. In conclusion, a small and transient reduction was found of the uptake of vitamin A into the lymph of TCDD-treated rats. It is obvious that this finding cannot explain the TCDD-induced decrease in hepatic vitamin A levels in the rat. Rather, a combination of inhibited retinol esterification in hepatic stellate cells, increased release of endogenous vitamin A, and increased hepatic catabolism of retinoids could explain the effect of TCDD on liver retinoid levels.
Asunto(s)
Contaminantes Ambientales/toxicidad , Absorción Intestinal/efectos de los fármacos , Linfa/metabolismo , Dibenzodioxinas Policloradas/toxicidad , Vitamina A/farmacocinética , Aciltransferasas/metabolismo , Administración Oral , Animales , Citocromo P-450 CYP1A1/metabolismo , Esterificación , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Hígado/enzimología , Masculino , Ratas , Ratas Sprague-Dawley , Retinoides/análisis , Retinol O-Graso-Aciltransferasa , Tritio , Vitamina A/administración & dosificaciónRESUMEN
Retinol absorbed and generated from dietary beta-carotene can be esterified by retinol esterifying enzyme(s) in intestinal absorptive cells. In this study, we observed the developmental changes and villus-crypt distribution of the activities of two retinol esterifying enzymes (lecithin-retinol acyltransferase (LRAT); and acyl-CoA-retinol acyltransferase (ARAT) in chick duodenum) to seek the possibility that these enzymes play distinct roles in retinol absorption and metabolism. Intestinal LRAT activity was barely expressed in embryonic stages until 2-3 d before hatching, when its activity becomes detectable; thereafter it abruptly increased to the maximal level at the third day of the posthatch period. In contrast, ARAT activity was present in the duodenum at the earliest stage examined, the 15th day of embryogenesis, and was elevated to the maximal level 3-4 d after hatching. An assay of LRAT and ARAT activities along the villus-crypt axis of the duodenum by a cryostat sectioning technique revealed that between the day of hatching and 1 d posthatch, an abrupt induction of LRAT activity occurred only in the villus region of the duodenum, where a coordinated induction of cellular retinol-binding protein, type II (CRBPII), was observed. In contrast, the rise in ARAT activity observed around the hatching period occurred at the broader portions of the villi including the area of villus-crypt junction. These observations in the developmental changes and distribution of LRAT and ARAT activities suggest that LRAT activity but not ARAT activity is closely related to the induction of CRBPII in the duodenum of developing chicks.