RESUMEN
Studies involving the cloning and disruption of the gene for acyl-CoA:diacylglycerol acyltransferase (DGAT) have shown that alternative mechanisms exist for triglyceride synthesis. In this study, we cloned and characterized a second mammalian DGAT, DGAT2, which was identified by its homology to a DGAT in the fungus Mortierella rammaniana. DGAT2 is a member of a gene family that has no homology with DGAT1 and includes several mouse and human homologues that are candidates for additional DGAT genes. The expression of DGAT2 in insect cells stimulated triglyceride synthesis 6-fold in assays with cellular membranes, and DGAT2 activity was dependent on the presence of fatty acyl-CoA and diacylglycerol, indicating that this protein is a DGAT. Activity was not observed for acyl acceptors other than diacylglycerol. DGAT2 activity was inhibited by a high concentration (100 mm) of MgCl(2) in an in vitro assay, a characteristic that distinguishes DGAT2 from DGAT1. DGAT2 is expressed in many tissues with high expression levels in the liver and white adipose tissue, suggesting that it may play a significant role in mammalian triglyceride metabolism.
Asunto(s)
Aciltransferasas/clasificación , Aciltransferasas/genética , Células 3T3 , Aciltransferasas/química , Tejido Adiposo/metabolismo , Secuencia de Aminoácidos , Animales , Northern Blotting , Diferenciación Celular , Línea Celular , Células Cultivadas , Clonación Molecular , ADN Complementario/metabolismo , Bases de Datos como Asunto , Diacilglicerol O-Acetiltransferasa , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Insectos , Cinética , Hígado/metabolismo , Cloruro de Magnesio/farmacología , Ratones , Datos de Secuencia Molecular , Mortierella/enzimología , Familia de Multigenes , Filogenia , Unión Proteica , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular , Triglicéridos/biosíntesisRESUMEN
In mice, the yolk sac appears to play a crucial role in nourishing the developing embryo, especially during embryonic days (E) 7;-10. Lipoprotein synthesis and secretion may be essential for this function: embryos lacking apolipoprotein (apo) B or microsomal triglyceride transfer protein (MTP), both of which participate in the assembly of triglyceride-rich lipoproteins, are apparently defective in their ability to export lipoproteins from yolk sac endoderm cells and die during mid-gestation. We therefore analyzed the embryonic expression of apoB, MTP, and alpha-tocopherol transfer protein (alpha-TTP), which have been associated with the assembly and secretion of apoB-containing lipoproteins in the adult liver, at different developmental time points. MTP expression or activity was found in the yolk sac and fetal liver, and low levels of activity were detected in E18.5 placentas. alpha-TTP mRNA and protein were detectable in the fetal liver, but not in the yolk sac or placenta. Ultrastructural analysis of yolk sac visceral endoderm cells demonstrated nascent VLDL within the luminal spaces of the rough endoplasmic reticulum and Golgi apparatus at E7.5 and E8.5. The particles were reduced in diameter at E13.5 and reduced in number at E18.5;-19. The data support the hypothesis that the yolk sac plays a vital role in providing lipids and lipid-soluble nutrients to embryos during the early phases (E7;-10) of mouse development. secretion in mouse yolk sac during embryonic development.
Asunto(s)
Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Saco Vitelino/embriología , Saco Vitelino/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN Complementario , Desarrollo Embrionario y Fetal/fisiología , Femenino , Expresión Génica , Immunoblotting , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , ARN/análisis , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Distribución Tisular/genética , Saco Vitelino/ultraestructuraAsunto(s)
Aldosterona/sangre , Glycyrrhiza , Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Mineralocorticoides/metabolismo , Plantas Medicinales , Renina/sangre , 11-beta-Hidroxiesteroide Deshidrogenasas , Anciano , Desoxicorticosterona/sangre , Enfermedades Transmitidas por los Alimentos/metabolismo , Ácido Glicirretínico/orina , Humanos , Hidrocortisona/sangre , Hidrocortisona/orina , Hipertensión/etiología , Hipopotasemia/etiología , Masculino , Desequilibrio Hidroelectrolítico/etiologíaRESUMEN
Rat adipocytes were treated with antisense dimethoxytrityl pentadecadeoxynucleotides, complementary to mRNA initiation codon regions for alpha and beta isozymes of protein kinase C (PKC). This antisense treatment provoked 50-70% decreases in PKC and insulin-stimulated 2-deoxyglucose uptake, but did not inhibit insulin-stimulated diacylglycerol synthesis. Sense or nonsense oligodeoxynucleotides were without effect on PKC and 2-deoxyglucose uptake. These results suggest that: (i) PKC-alpha and PKC-beta isozymes can be specifically downregulated in rat adipocytes by antisense oligodeoxynucleotides, and (ii) insulin-stimulated glucose transport requires PKC.