RESUMEN
In vivo, refeeding starved chickens stimulates transcription of the avian gene for malic enzyme in liver; in hepatocytes in culture, triiodothyronine (T3) and insulin stimulate transcription of this gene. In vivo, starvation, and in hepatocytes in culture, glucagon, medium-chain fatty acids (MCFA) and long-chain fatty acids (LCFA) inhibit transcription of the malic enzyme gene. We have defined a T3-response unit in the 5'-flanking DNA of the malic enzyme gene; it contains one major T3 response element and several minor ones; maximum responsiveness is dependent on the presence of all of these elements. LCFA probably act by inhibiting binding of T3 to its nuclear receptor. MCFA appear to act by a different mechanism. Inhibitory MCFA have chain lengths of six, seven or eight carbons; a common feature of other inhibitory compounds is that they can be metabolized to MCFA. Eight-carbon fatty acids with a hydroxyl on the 2- or 3-carbon are more potent inhibitors than octanoate, whereas 2-bromo-fatty acids and 2-hydroxy hexanoate are not inhibitory. In transfection experiments with a large variety of constructs derived from the malic enzyme 5'-flanking DNA, the ability of fatty acids to inhibit promoter function localizes to regions of DNA that contain T3REs. Promoter function of artificial T3REs also is inhibited by MCFA. Inhibition of promoter function using malic enzyme DNA is relatively constant in magnitude irrespective of the size of the T3 response. We postulate that MCFA directly regulates one of the functions of the T3 receptor.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Hígado/enzimología , Malato Deshidrogenasa/biosíntesis , Transcripción Genética , Triyodotironina/farmacología , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Sitios de Unión , Células Cultivadas , Pollos , Ácidos Grasos no Esterificados/farmacología , Glucagón/farmacología , Hígado/efectos de los fármacos , Malato Deshidrogenasa/genética , Proteínas Recombinantes/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Eliminación de Secuencia , Inanición , Transcripción Genética/efectos de los fármacosRESUMEN
In vivo, feeding stimulates and starvation inhibits transcription of the malic enzyme gene. In chick-embryo hepatocytes in culture, triiodothyronine (T3) stimulates and glucagon inhibits transcription of this gene. As a first step in the characterization of the involved regulatory mechanisms, fragments of genomic DNA spanning the structural and 5'-flanking regions of the chicken malic enzyme gene were cloned. The coding region of the gene is organized into 14 exons and 13 introns and is greater than 106 kb in length. The size of the gene, the number and lengths of the exons, and positions at which introns are inserted into the coding regions are virtually identical in the chicken and rat genes. When transiently transfected into chick-embryo hepatocytes, 5800 bp of 5'-flanking DNA conferred T3 responsiveness to a linked chloramphenicol acetyltransferase (CAT) reporter gene. Using deletion and site-specific mutations of 5'-flanking DNA, we identified a complex T3 response unit that contains one major T3 response element (T3RE) and several minor ones. The major element contains two degenerate copies of the hexamer, RGGWMA, separated by 4 bp and was a strong repressor in the absence of ligand. Endogenous levels of T3 receptor are sufficient to allow the T3 response elements in the upstream region of the malic enzyme gene to confer responsiveness to T3, suggesting that they are physiologically relevant.
Asunto(s)
Pollos/genética , Malato Deshidrogenasa/biosíntesis , Malato Deshidrogenasa/genética , Secuencias Reguladoras de Ácidos Nucleicos , Triyodotironina/farmacología , Animales , Secuencia de Bases , Células Cultivadas , Embrión de Pollo , Cloranfenicol O-Acetiltransferasa/biosíntesis , Hígado/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos/efectos de los fármacos , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
We have provided a historical and personal description of the analysis of physiological and molecular mechanisms by which diet and hormones regulate the activity of hepatic malic enzyme. For the most part, our analyses have been reductionist in approach, striving for increasingly simpler systems in which we can ask more direct questions about the molecular nature of the signaling pathways that regulate the activity of malic enzyme. The reductionist approaches that were so successful at analyzing molecular mechanisms in cells in culture may now provide the means to analyze more definitively questions about the physiological mechanisms involved in nutritional regulation of gene expression. In addition to physiological questions, however, there are still many aspects of the molecular mechanisms that have not been elucidated. Despite considerable effort from many laboratories, the molecular mechanisms by which T3 regulates transcription are not clear. Similarly, the molecular details for the mechanisms by which glucagon, insulin, glucocorticoids, and fatty acids regulate gene expression remain to be determined. The role of fatty acids is particularly interesting because it may provide a model for mechanisms by which genes are regulated by metabolic intermediates; this is a form of transcriptional regulation widely used by prokaryotic organisms and extensively analyzed in prokaryotic systems, but poorly understood in higher eukaryotes. At any specific time, there is, of course, only one rate of transcription for each copy of the malic-enzyme gene in a cell. Our long-term objective is to understand how signals from all of the relevant regulatory pathways are integrated to bring about that rate.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Malato Deshidrogenasa/genética , Animales , Hormonas/fisiología , Fenómenos Fisiológicos de la Nutrición/fisiologíaRESUMEN
The response of maternal plasma calcium concentration to the abrupt and permanent removal of the suckling pups on Day 13 of lactation was investigated. Maternal plasma calcium did not change at 6 hr or 12 hr following pup removal. At 18 hr and 24 hr after weaning, the maternal plasma calcium concentration increased in mothers consuming either 0.47% calcium or 0.02% calcium diets. At 24 hr after weaning, the plasma calcium increase in mothers consuming low dietary calcium was 55% that of mothers consuming adequate dietary calcium. The contribution of the mammary gland to the plasma calcium increase in rats consuming the low dietary calcium was investigated by removing the mammary glands. Following mammary gland removal, plasma calcium increased 50% compared with mothers that had intact mammary glands. The data suggest that intestinal absorption of calcium and bone calcium mobilization remain stimulated by the lactation process for at least 24 hr after removal of the nursing pups.
Asunto(s)
Huesos/metabolismo , Calcio/sangre , Mucosa Intestinal/metabolismo , Glándulas Mamarias Animales/metabolismo , Destete , Animales , Calcio/metabolismo , Calcio de la Dieta/administración & dosificación , Femenino , Lactancia/metabolismo , Embarazo , RatasRESUMEN
The requirement of parathyroid tissue for bone mineral loss during lactation was investigated. Lactating rats parathyroidectomized (PTX) at day 2 of lactation and consuming a 2% calcium diet are hypercalcemic and hypophosphatemic at day 13 of lactation. The high-calcium diet supports normal growth of pups nursing PTX mothers. PTX lactating rats mobilize bone mineral to the same extent as euparathyroid lactating rats consuming the same diet. Non-lactating PTX rats lose no bone mineral over a similar time period, indicating lactation-specific bone mineral mobilization in the absence of parathyroid tissue. PTX rats were verified to have physiologically insignificant amounts of parathyroid tissue, as evidenced by severe hypocalcemia and/or death in each rat after a shift from a 2% calcium to a 0.02% calcium diet. These results conclusively demonstrate that lactation-associated bone mineral mobilization does not require parathyroid hormone or parathyroid tissue.
Asunto(s)
Densidad Ósea , Lactancia/fisiología , Hormona Paratiroidea/deficiencia , Animales , Animales Recién Nacidos , Peso Corporal , Calcio/sangre , Femenino , Fémur/anatomía & histología , Tamaño de los Órganos , Concentración Osmolar , Paratiroidectomía , Fósforo/sangre , Embarazo , Ratas , Ratas EndogámicasRESUMEN
The effectiveness of a combination of 1 alpha-hydroxyvitamin D3 and 25-hydroxyvitamin D3 for reducing incidence of parturient paresis in aged Holstein cows was tested. Intramuscular injection of .5 mg of 1 alpha-hydroxyvitamin D3 plus 4 mg of 25-hydroxyvitamin D3 increased plasma 1,25-dihydroxyvitamin D concentrations through parturition. Treatment with 1 alpha-hydroxyvitamin D3 plus 25-hydroxyvitamin D3 raised prepartum serum Ca approximately 2 mg/dl and prepartum serum P approximately 4 to 5 mg/dl higher than untreated controls. Both treated and control cows had approximately a 2-mg/dl decrease in serum Ca following parturition. The prepartum diet of alfalfa silage and hay was supplemented with a grain mixture supplying 100 g of Ca/d from ground limestone. Under these dietary conditions, incidence of parturient paresis was reduced from 33 to 8%. In a separate experiment, treatment with 1 alpha-hydroxyvitamin D3 plus 25-hydroxyvitamin D3 did not reduce incidence of parturient paresis when cows consumed mixed diets of different feed-stuff composition. Further experiments are required to determine specifically the factor or factors responsible for the difference in response to active vitamin D compound administration between the two experiments. Prepartum dietary Ca intake may be one such factor.
Asunto(s)
Calcifediol/uso terapéutico , Calcio de la Dieta/administración & dosificación , Enfermedades de los Bovinos/prevención & control , Hidroxicolecalciferoles/uso terapéutico , Parálisis de la Parturienta/prevención & control , Animales , Calcifediol/administración & dosificación , Calcitriol/sangre , Calcio/administración & dosificación , Calcio/sangre , Calcio de la Dieta/farmacocinética , Bovinos , Combinación de Medicamentos , Femenino , Hidroxicolecalciferoles/administración & dosificación , Inyecciones Intramusculares/veterinaria , Absorción Intestinal/efectos de los fármacos , Trabajo de Parto/sangre , Fósforo/sangre , EmbarazoRESUMEN
Fifty percent (7/14) of aged cows treated with 4 mg 24,25-dihydroxyvitamin D3 intramuscularly precalving developed parturient paresis shortly after calving compared with 7% (1/14) of controls. Injection of 24,25-dihydroxyvitamin D3 increased concentrations in blood plasma 15 times that in control cows. Blood plasma 1,25-dihydroxyvitamin D concentrations were elevated in all groups on day of calving but were not different. Injection of 24,25-dihydroxyvitamin D3 did not alter the typical plasma profile for calcium, phosphorus, or 1,25-dihydroxyvitamin D of paretic or nonparetic cows around parturition. Although injection of 24,25-dihydroxyvitamin D3 was associated with an increased incidence of parturient paresis, the mechanism remains unknown.