RESUMEN
Development of the follicle in egg-laying species such as the chicken is regulated by systemic factors as well as by the highly orchestrated interplay of differentially expressed genes within this organ. Differential mRNA display analysis of defined phases of follicle development resulted in the characterization of coagulation factor XIIIA. It is expressed and produced by cells of the theca externa in a highly regulated manner during distinct growth phases of the follicle. Transcripts for factor XIIIA are already detectable at the beginning of follicle development and peak at the end of phase 2. Protein levels, however, still increase during phase 3, peak shortly after ovulation, and persist until the postovulatory tissue is completely resorbed. Factor XIIIA is secreted as a monomer into the extracellular matrix of the theca externa and is not associated with factor XIIIB as is the case in plasma. Our data suggest that, due to its transglutaminase activity, factor XIIIA stabilizes the follicular wall by cross-linking matrix components. Thus, coagulation factor XIIIA might play a key role in coping with the massive mechanical stress exerted by the large amount of yolk accumulating during the rapid growth phase of the oocyte.
Asunto(s)
Folículo Ovárico/metabolismo , Células Tecales/metabolismo , Transglutaminasas/biosíntesis , Secuencia de Aminoácidos , Animales , Membrana Basal/metabolismo , Northern Blotting , Western Blotting , Cloruro de Calcio/farmacología , Línea Celular , Pollos , ADN Complementario/metabolismo , Ácido Edético/farmacología , Femenino , Perfilación de la Expresión Génica , Glutatión Transferasa/metabolismo , Células de la Granulosa/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Ovulación/fisiología , Pruebas de Precipitina , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transfección , Transglutaminasas/metabolismoRESUMEN
In the current studies we describe the effects of PD 72953 and related compounds on lipoprotein levels in chow-fed male rats. After 2 weeks, 10 mg/kg of PD 72953 daily was as effective as 100 mg/kg gemfibrozil for elevating HDL-cholesterol. At 100 mg/kg, PD 72953 further elevated HDL-cholesterol to 232% of control levels, and was associated with increased HDL size and plasma apoE (169% of control), despite no change in hepatic apoE mRNA. ApoA-I rose transiently (at 1 week), but by 2 weeks only apoE remained elevated. PD 72953 dose-dependently reduced plasma apoB, VLDL-cholesterol, LDL-cholesterol, and triglyceride. Hepatic apoC-III mRNA reduction parallelled triglyceride lowering. After 1 week, 30 and 100 mg/kg per day PD 72953 reduced plasma apo-CIII levels by 30 and 34%, and triglycerides by 60 and 83%, respectively. PD 72953 treatment had no effect on triglyceride production rates; however, 125I-labeled VLDL apoB disappearance was enhanced. We compared PD 72953 to a structurally similar diacid, PD 69405, that also reduced VLDL and LDL, but had no effect on HDL elevation. Compared to PD 72953, PD 69405 further accelerated 125I-labeled VLDL apoB disappearance, decreased triglyceride production, and elevated the ratio of post-heparin hepatic to lipoprotein lipase activity. Whole animal studies, transient transfection studies in HepG2 cells, and chimeric receptor studies in kidney 293 cells suggest that PD 72953 is a ligand for the peroxisomal proliferation activated receptor alpha (PPARalpha), and PPARgamma. Overall, PD 72953 may act through a peroxisomal proliferation activated receptor and result in plasma triglycerides and apoB-containing lipoprotein reduction, while also raising HDL cholesterol. Reduced apoC-III may allow triglyceride-rich remnants to more efficiently bind and present substrate to peripheral tissue lipoprotein lipase, and therefore allow enhanced shedding of remnant phospholipid surface for HDL production.
Asunto(s)
Caproatos/farmacología , HDL-Colesterol/sangre , Hipolipemiantes/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Apolipoproteína C-III , Apolipoproteínas B/sangre , Apolipoproteínas C/sangre , Apolipoproteínas C/genética , Apolipoproteínas E/sangre , Caproatos/síntesis química , Caproatos/metabolismo , LDL-Colesterol/sangre , VLDL-Colesterol/sangre , Gemfibrozilo/farmacología , Humanos , Hígado/metabolismo , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Triglicéridos/sangreRESUMEN
The HNF-4 orphan receptor is a member of the nuclear receptor family of transcription factors and a major regulator of genes involved in carbohydrate and lipid metabolism. As an initial step in characterizing the role of HNF-4 in the regulation of metabolism, we have generated a dominant negative form of HNF-4 (DN-HNF-4) that contains a defective DNA-binding domain. In gel mobility shift assays, DN-HNF-4 did not bind an oligonucleotide probe representing an essential HNF-4 binding site, C3P contained in the human apo CIII promoter, but did prevent the binding of two recombinant isoforms, HNF-4alpha1 and HNF-4alpha2, as well as naturally-occurring HNF-4. DN-HNF-4 had no effect on the binding of PPARgamma-RXRalpha heterodimers to a PPAR response element. In transfected HepG2 cells, DN-HNF-4 dramatically reduced constitutive transcriptional activity of the human apo CIII promoter and abolished the positive transcriptional activity caused by plasmids expressing either isoform of HNF-4. These results indicate that DN-HNF-4 is a selective dominant negative mutant which forms defective heterodimers with wild-type HNF-4, thereby preventing DNA binding and subsequent transcriptional activation by HNF-4.
Asunto(s)
Proteínas de Unión al ADN/genética , Mutación , Fosfoproteínas/genética , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Apolipoproteína C-III , Apolipoproteínas C/genética , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Expresión Génica , Genes Reporteros , Factor Nuclear 4 del Hepatocito , Humanos , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Transcripción Genética , Dedos de ZincRESUMEN
For the last 30 years fibrates have been widely prescribed to treat human dyslipidemia. However, the primary mechanism by which they lower plasma lipid levels is still unknown. Studies with transgenic mice have suggested that changes in apoC-III expression levels have a dramatic influence on plasma triglyceride levels. These results suggested that fibrates could reduce lipid levels by lowering apoC-III gene expression. In the current studies, we sought to determine whether the selected fibrates, bezafibrate, clofibrate, fenofibrate, and gemfibrozil, could reduce hepatic apoC-III mRNA and plasma apoC-III levels. Chow-fed rats were orally gavaged daily with a dosing vehicle alone or with 100 mg/kg of each of the fibrates for 1 week and in addition with gemfibrozil for 2 weeks. Bezafibrate and fenofibrate lowered plasma triglyceride by approximately half and dramatically reduced hepatic apoC-III mRNA and plasma apoC-III levels. In contrast, clofibrate did not reduce plasma triglyceride levels and only partially reduced apoC-III mRNA and plasma protein levels. Gemfibrozil strongly reduced plasma triglyceride levels and had an intermediate but significant effect on apoC-III mRNA and plasma apoC-III levels. Some of the fibrates, especially gemfibrozil also reduced plasma apoC-II levels, an effect that could contribute to the observed triglyceride-lowering effect. In addition, the ratio of plasma apoE to plasma apoC-II plus apoC-III was strongly and inversely correlated with plasma triglyceride levels. As plasma apoE levels were not reduced in gemfibrozil-treated animals, this could also have contributed to the triglyceride-lowering effect of this fibrate. Fibrate-mediated triglyceride lowering was not the result of a decreased apoB or VLDL production and, therefore, suggested an enhanced VLDL remnant catabolism. Our results suggest that the mechanism by which fibrates lower plasma triglycerides is by reducing the level of hepatic apoC-III expression.
Asunto(s)
Apolipoproteínas C/metabolismo , Hipolipemiantes/farmacología , Hígado/efectos de los fármacos , Animales , Apolipoproteína C-III , Apolipoproteínas C/genética , Bezafibrato/farmacología , Colesterol/sangre , Clofibrato/farmacología , Fenofibrato/farmacología , Gemfibrozilo/farmacología , Hígado/metabolismo , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Triglicéridos/sangreRESUMEN
We have characterized the molecular defect causing lecithin:cholesterol acyltransferase (LCAT)-deficiency (LCAT-D) in the LCAT gene in three siblings of Austrian descent. The patients presented with typical symptoms including corneal opacity, hemolytic anemia, and kidney dysfunction. LCAT activities in the plasma of these three patients were undetectable. DNA sequence analysis of polymerase chain reaction (PCR)-amplified DNA of all six LCAT exons revealed a new point mutation in exon IV of the LCAT gene, i.e., a G to A substitution in codon 140 converting Arg to His. This mutation caused the loss of a cutting site for the restriction endonuclease HhaI within exon IV: Upon digestion of a 629-bp exon IV PCR product with HhaI, the patients were found to be homozygous for the mutation. Eight of 11 family members were identified as heterozygotes. Transfection studies of COS-7 cells with plasmids containing a wild-type or a mutant LCAT cDNA revealed that, in contrast to the cell medium containing wild-type enzyme, no enzyme activity was detectable upon expression of the mutant protein. This represents strong evidence for the causative nature of the observed mutation for LCAT deficiency in affected individuals and supports the conclusion that Arg140 is crucial for the structure of an enzymatically active LCAT protein.
Asunto(s)
Exones , Fosfatidilcolinas/genética , Esterol O-Aciltransferasa/deficiencia , Esterol O-Aciltransferasa/genética , Adenosina , Arginina , Secuencia de Bases , Femenino , Expresión Génica/genética , Guanosina , Histidina , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Linaje , Mutación Puntual , Reacción en Cadena de la PolimerasaRESUMEN
A previously undescribed single missense mutation (C-->G) was detected within exon 5 of the LPL gene in two members of an Italian family affected with type I hyperlipoproteinemia. This mutation causes a highly conservative amino acid replacement (Asp-->Glu) at position 180 of the mature LPL protein resulting in a virtual absence of LPL enzyme activity and LPL enzyme mass in postheparin plasma. Adipose tissue mRNA concentrations and mRNA sizes were not affected. Both patients were homozygous for the mutation, whereas the parents were heterozygous. Comparison of the expression of the mutated cDNA and the wildtype cDNA in cos-7 cells revealed proper transcription and translation of the mutated clone into an immunologically detectable protein. The mutated LPL protein was secreted from the cells in a manner similar to that of wild-type LPL and bound to heparin-Sepharose with identical properties. However, the mutated enzyme, in contrast to wildtype LPL, exhibited no detectable lipolytic activity against a triglyceride substrate. Our results demonstrate that even a highly conservative amino acid replacement outside the proposed active site of LPL is incompatible with proper enzyme function.