RESUMEN
The regulation of transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (4.1.1.32) during diabetes is a complex process that involves a number of regulatory elements in the PEPCK-C gene promoter. The accessory factor 2 (AF2)-binding region that is contained within the glucocorticoid regulatory unit of the PEPCK-C gene promoter (-451 to -353) has been implicated in the action of both insulin and glucocorticoids on PEPCK-C gene transcription. To determine the role of AF2 in these processes, we have generated a mouse model bearing a transgene that contains the PEPCK-C gene promoter with a mutation in the AF2-binding region. This promoter is linked to the structural gene for human growth hormone that is biologically inactive (AF2-2000/hGx). In the absence of the AF2 regulatory element, the transcription of the transgene in the liver is not induced by diabetes but is inhibited by the administration of insulin. There is also a marked reduction in the response of the AF2-2000/hGx gene in the kidney to the administration of glucocorticoids. The AF2-2000/hGx gene in the liver responds normally to a high carbohydrate diet with a marked decrease in gene transcription. This suggests that insulin is not exerting its usual negative effect on the PEPCK-C gene promoter through the AF2 site. In contrast, the response of this transgene to a high fat/carbohydrate-free diet is severely blunted. Our results support a role for the AF2 site in the PEPCK-C gene promoter in the effect of glucocorticoids, but not insulin, on PEPCK-C gene transcription in the liver.
Asunto(s)
Diabetes Mellitus Experimental/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Animales , Secuencia de Bases , Cartilla de ADN , Ratones , Ratones Transgénicos , Secuencias Reguladoras de Ácidos Nucleicos , TransgenesRESUMEN
Modern genetics has developed methods to modify the expression of genes in animals to study the factors responsible for the tissue-specific expression and hormonal and dietary regulation of metabolic processes. As these methods are applied to genes that code for critical proteins in metabolic pathways, a new insight into the control of metabolism is emerging. There are three general approaches currently in use. First, is the introduction of genes into the germ line to create transgenic animal models in which the gene of interest is over-expressed. This model is of particular value for promoter analysis because it is possible to introduce specific mutations into a putative regulatory region of a transgene and study its transcriptional control in the intact animal. Second, the developmental function of a gene product and its effect on various metabolic processes in a mouse can be directly determined by deleting a gene of interest by homologous recombination. Gene "knockout" mice are currently available with deletions in the genes for a variety of transcription factors and other biologically active proteins, permitting a critical analysis of the proteins responsible for the metabolic patterning of the animal. Third, the metabolic role of a gene of interest in a specific tissue can be studied by ablating its mRNA by the introduction of a transgene that codes for antisense mRNA targeted against the gene transcript. Because it is possible to use a transgene with a tissue-specific promoter, this procedure allows the isolation of the metabolic effect to a selected tissue in the transgenic animal. Taken together, these procedures provide a unique set of metabolic models for an in-depth study of metabolic regulation. This review will present examples of selected animal models currently available and will outline the challenge these animals present for investigators in the nutritional sciences.
Asunto(s)
Metabolismo Energético/genética , Ratones Transgénicos/metabolismo , Modelos Biológicos , Animales , Secuencia de Bases , ADN/análisis , ADN/química , ADN/genética , Metabolismo Energético/fisiología , Eliminación de Gen , Regulación de la Expresión Génica , Hígado/fisiología , Ratones , Ratones Transgénicos/fisiología , Datos de Secuencia Molecular , Fosfoenolpiruvato Carboxiquinasa (ATP) , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/fisiología , ARN Mensajero/análisis , ARN Mensajero/química , ARN Mensajero/genética , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Transcripción GenéticaRESUMEN
Identification of biochemical changes induced by ethanol ingestion would aid in the diagnosis and management of many alcohol-related problems in man. In this paper we identify a pH 5.5 alpha-mannosidase activity in the rat which is affected by chronic ethanol consumption. Chronic (16 wk) ingestion of alcohol (36% of calories) causes the activity of this alpha-mannosidase (thought to be the cytosolic alpha-mannosidase) in liver to decrease by 50%. We hypothesize that this deficiency of (pH 5.5) alpha-mannosidase activity may account for the reduced rate of secretion of glycoproteins by livers of alcohol-fed rats reported by other investigators (Volentine et al, Hepatology 1987;7:490-495).