RESUMEN
Expression of the oncodevelopmental alpha-fetoprotein (AFP) gene is tightly regulated and occurs in the yolk sac, fetal liver and intestine, and cancerous liver cells. Transcription of the AFP gene is under the control of three enhancers that are very tissue specific. We have shown that the most upstream of these enhancers, located at -6 kb, works through the combined action of liver-enriched factors and nuclear receptors that bind to three regions of this DNA regulatory element. This study showed that orphan nuclear receptors of the ROR alpha, Re-verb alpha, and Rev-erb beta groups can bind as monomers with high affinity and specificity to an evolutionarily conserved AGGTCA motif in the functionally important region 1 of this AFP enhancer. Transient transfection experiments performed with human HepG2 hepatoma cells showed that overproduction of ROR alpha 4 stimulated the activity of the AFP enhancer in a dose-dependent manner, while that of Rev-erb alpha and Rev-erb beta had the opposite effect. These effects were highly specific and required the integrity of the AGGTCA motif. The action of these nuclear receptors also occurred in the context of the entire 7-kb regulatory region of the rat AFP gene. These results suggest that altering the amounts or activities of these orphan receptors in cells of hepatic or endodermal origin could modulate AFP gene expression in response to a variety of developmental or carcinogenic stimuli.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Hormona Tiroidea , alfa-Fetoproteínas/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Células CACO-2 , Pollos , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Humanos , Ratones , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Plásmidos , Unión Proteica , Proteínas/genética , Proteínas/metabolismo , ARN/genética , ARN/metabolismo , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genética , Transactivadores/metabolismo , Transfección , Células Tumorales Cultivadas , alfa-Fetoproteínas/genéticaRESUMEN
The alpha-fetoprotein (AFP) gene is expressed mainly in the yolk sac, liver and intestine during embryonic and fetal life. We have analyzed the activities of some of the rat AFP regulatory elements in vivo, using transgenic mice bearing the LacZ gene with a nuclear localization signal (nls-lacZ) placed under the control of the rat AFP promoter and the most proximal enhancer regions (from -3,127 to +102). Four of the six transgenic lines, with two genetic backgrounds, had highly specific reproducible patterns of transgene expression on embryonic days E10.5, E12 and E15. Analyses were performed on the whole embryo and histologically. There was nuclear staining in the yolk sac endodermal cells and in the epithelial cells of the intestine, indicating that the proximal enhancer and promoter drive expression in these cells where the AFP gene is actively transcribed. The pharyngo-tympanic canal was also stained in the transgenic embryos. But there was no expression of the lacZ transgene in the embryonic liver, indicating that additional sequences of rat genomic DNA are required for correct expression in the liver.
Asunto(s)
Elementos de Facilitación Genéticos , Trompa Auditiva/metabolismo , Mucosa Intestinal/metabolismo , Regiones Promotoras Genéticas , Saco Vitelino/metabolismo , alfa-Fetoproteínas/genética , Animales , Expresión Génica , Genes Reporteros , Ratones , Ratones Transgénicos , RatasRESUMEN
Alpha-foetoprotein (AFP), the major plasma protein in the foetus, is mainly synthesized by yolk sac and foetal liver. It binds polyunsaturated fatty acids and probably controls their metabolism and action. We investigated the effects of fatty acids and fibrates on expression of the AFP gene using two complementary approaches. Treatment with 5-8-11-14 eicosatetraynoic acid (ETYA), an analogue of arachidonic acid, specifically led to lower AFP mRNA levels in cultured rat yolk sac explants whereas treatment with palmitic or oleic acid did not. Clofibric acid and fenofibrate also gave lower AFP mRNA levels. Transient transfection experiments with HepG2 hepatoma cells showed that ETYA and clofibric acid decreased the transcriptional activity of the 7 kb regulatory region of the rat AFP gene. The 330 bp AFP promoter was identified as a target for these down regulating effects.
Asunto(s)
Ácido Clofíbrico/farmacología , Ácidos Grasos/farmacología , Fenofibrato/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , alfa-Fetoproteínas/genética , Ácido 5,8,11,14-Eicosatetrainoico/farmacología , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Técnicas de Cultivo , Regulación hacia Abajo/genética , Femenino , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Ratas Wistar , Transfección , Células Tumorales Cultivadas , Saco Vitelino , alfa-Fetoproteínas/biosíntesisRESUMEN
The visceral yolk sac is a fetal membrane with essential placental functions. It is the major site of synthesis of alpha-fetoprotein (AFP), the most abundant plasma protein in the fetus. We developed a system of rat yolk sac explants in serum-free culture medium to study the regulation of endodermal gene expression in yolk sac. The explanted yolk sac tissues retained their double-sided morphology for up to 48 hours. The epithelial cells of both layers remained tightly joined on a basement membrane as seen by light and electron microscopy. This probably accounts for the continued expression of several endodermal cell-specific markers. The levels of mRNA encoding AFP, vitamin D-binding protein (DBP), hepatocyte nuclear factor 1alpha and beta transcription factors did not change during the 48-hour culture period. This reflects the stability of the differentiation state of the yolk sac endodermal cells. Dexamethasone and phorbol ester (TPA) specifically reduced the AFP mRNA level without affecting that of DBP. This suggests that these transduction pathways are functional in the yolk sac during this period of gestation and could be involved in the physiological down-regulation of AFP gene expression before birth. All these results show that this serum-free culture of rat yolk sac explants is a valuable system for further investigating the action of natural compounds and pharmacological drugs on endodermal gene expression during the embryonic and fetal periods.
Asunto(s)
Dexametasona/farmacología , Regulación hacia Abajo/genética , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Saco Vitelino/metabolismo , alfa-Fetoproteínas/genética , Animales , Técnicas de Cultivo , Regulación hacia Abajo/efectos de los fármacos , Endodermo/citología , Femenino , Masculino , Ratas , Ratas Wistar , Saco Vitelino/citologíaRESUMEN
The effects of phorbol esters (phorbol-12,13-dibutyrate, PDB) on alpha-fetoprotein expression and cell growth were assayed by using fetal hepatocytes in primary culture. PDB acts synergistically with epidermal growth factor (EGF) to specifically decrease alpha-fetoprotein (AFP) mRNA levels, without affecting the expression of other genes of the same family, such as albumin and Vitamin D-binding protein (DBP). This effect is PDB-dose dependent, maximal effects being at 10 ng/ml. The implication of protein kinase C (PKC) in this effect seems clear since bisindolylmaleimide (BIS), a specific PKC inhibitor, completely blocks the PDB effect on AFP expression. Nuclear run-on experiments show that the decrease in AFP mRNA levels is mainly due to an inhibition in the transcription rate of the gene. Determination of PKC activities shows that fetal hepatocytes contain mainly Ca(2+)-independent isoenzymes, which patterns of activation was not modified by EGF plus PDB treatment with respect to PDB treatment. We have found that MAPK and JNK activities, c-jun and c-fos mRNA levels and AP-1 binding activity are notably increased when cells are incubated with both EGF and PDB, PDB does not stimulate growth of fetal hepatocytes, measured either as [3H]-thymidine incorporation into DNA or by cell cycle analysis using flow cytometry. All these results suggest that activation of PKC may affect liver gene expression rather than cell growth in fetal hepatocytes.
Asunto(s)
Hígado/embriología , Hígado/fisiología , Proteínas Quinasas Activadas por Mitógenos , Forbol 12,13-Dibutirato/farmacología , alfa-Fetoproteínas/genética , Albúminas/efectos de los fármacos , Albúminas/genética , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo , Factor de Crecimiento Epidérmico/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes fos , Genes jun , Proteínas Quinasas JNK Activadas por Mitógenos , Hígado/citología , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/efectos de los fármacos , alfa-Fetoproteínas/efectos de los fármacosRESUMEN
We have further characterized the most distal of the three alpha-fetoprotein (AFP) enhancers required for expression of the AFP gene in fetal hepatocytes and yolk sac endodermal cells. Almost total rat AFP enhancer 3 (E3) activity is driven by a 160-bp fragment at -6 kb containing three target regions for nuclear proteins that cooperate to stimulate transcription from the AFP and the thymidine kinase promoters in HepG2 hepatoma cells. Region 1, recently shown to be crucial for correct function of the enhancer in liver of transgenic mice, is recognized by two sets of transcription factors that bind to partly overlapping sites, 1a and 1b, in a noncooperative and nonexclusive manner. Site 1a contains a motif, AGGTCA, which is recognized by chicken ovalbumin upstream promoter transcription factors (COUP-TFs), but not by hepatocyte nuclear factor 4. Hepatocyte nuclear factor 3 (HNF3) and CCAAT/enhancer binding protein (C/EBP), which bind to regions 2 and 3, respectively, are likely responsible for the liver-specific E3 action. They play a key role by acting in synergy. The participation of nuclear receptors such as COUP-TFs, with C/EBP and HNF3, in the tight control of the distal AFP enhancer is a new, and perhaps key, step toward understanding the regulation and function of this enhancer, which may remain active throughout development.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Factores de Transcripción/metabolismo , alfa-Fetoproteínas/genética , Animales , Proteínas Potenciadoras de Unión a CCAAT , Factor de Transcripción COUP I , Carcinoma Hepatocelular , Pollos , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor Nuclear 3-alfa del Hepatocito , Hígado/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Unión Proteica , Ratas , Células Tumorales CultivadasRESUMEN
The oncodevelopmentally regulated alpha-fetoprotein (AFP) gene offers a very good model system to better understand the molecular mechanisms which dictate the specificity of gene expression in liver and control its tight modulation in the course of development and carcinogenesis. Transcription factors of the CCAAT/enhance-binding protein (C/EBP), hepatocyte nuclear factor-1 (HNF-1), and nuclear factor-1 (NF-1) families can bind in vitro to the promoter of the rat AFP gene, which makes the expression of the AFP gene specific to the liver. We have evaluated the influence of some of these factors on the activity of the AFP promoter by transfection of HepG2 hepatoma cells with the appropriate expression vector plus a CAT plasmid under the control of the AFP promoter. A similar plasmid bearing the rat albumin promoter was used as a control. C/EBP alpha, and C/EBP beta acted as transactivators on the AFP promoter, while LIP, a truncated form of C/EBP beta, was a potent negative regulator of the promoter. Interestingly, HNF-1 beta was found to be more potent than HNF-1 alpha in activating the AFP promoter in the HepG2 cells. This effect was highly promoter and cell specific since it did not occur with the rat albumin promoter or in Chinese hamster ovary cells. HNF-1 beta, which is produced earlier than HNF-1 alpha during liver development, would thus have the greater influence on the AFP promoter in early development. Our results pointed to a key role that NF1 might play in the functioning of the AFP promoter. Indeed, overexpression of NF1 induced a specific decrease in the activity of the AFP promoter. Competition between NF1 and HNF-1 for binding to their overlapping binding sites on the AFP promoter would be critical for modulating its activity.
Asunto(s)
Hígado/fisiología , Regiones Promotoras Genéticas , Factores de Transcripción/genética , alfa-Fetoproteínas/genética , Animales , Proteínas Potenciadoras de Unión a CCAAT , Transformación Celular Neoplásica , Cricetinae , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Femenino , Regulación de la Expresión Génica , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Técnicas In Vitro , Masculino , Factores de Transcripción NFI , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ratas , Factores de Transcripción/metabolismo , Transfección , Células Tumorales Cultivadas , Proteína 1 de Unión a la Caja Y , alfa-Fetoproteínas/metabolismoRESUMEN
The effects of a phorbol ester (TPA) and of members of the Jun and Fos oncoprotein family on the activity of the rat alpha-fetoprotein (AFP) promoter were checked by using transient expression experiments in HepG2 hepatoma cells. TPA blocked the activity of the rat AFP promoter in a dose-dependent manner. Overexpression of c-Jun specifically repressed the rat AFP promoter but not the albumin promoter. JunB and JunD were poorer inhibitors. c-Fos expression did not potentiate the negative effect of Jun. The Jun-induced repression does not require binding of c-Jun to the AFP promoter. DNase 1 footprinting experiments did not display any high affinity binding site for Jun on the AFP promoter. Integrity of the c-Jun DNA binding domain is not required for the c-Jun protein to block the AFP promoter. The N-terminal part of Jun, which contains the activating domain, is responsible for the repression as shown by using Jun-Gal4 chimera. Jun likely exerts its negative control on the AFP promoter via protein-protein interactions with a not yet identified trans-activating factor within the -134 to +6 region or with a component of the general machinery of transcription. Jun proteins can thus be key intermediates in regulatory cascades which result in the differential modulation of the AFP and albumin gene expression in the course of liver development and carcinogenesis.
Asunto(s)
ADN/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/genética , alfa-Fetoproteínas/genética , Animales , Carcinoma Hepatocelular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Regulación de la Expresión Génica , Humanos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-jun/fisiología , Ratas , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
The promoter of the rat alpha-fetoprotein (AFP) gene, which makes the expression of the developmentally regulated AFP gene specific to the liver, is a putative target for transcription factors of the CAAT/enhancer-binding protein (C/EBP), hepatocyte nuclear factor-1 (HNF-1) and nuclear factor-1 (NF-1) families. We have evaluated the influence of these factors on the activity of the AFP promoter by transfection of HepG2 hepatoma cells with the appropriate expression vector plus a CAT plasmid under the control of the AFP promoter. A similar plasmid bearing the rat albumin promoter was used as a control. C/EBP alpha, C/EBP beta and D-binding protein (DBP) acted as trans-activators on the AFP promoter, whereas liver inhibitory protein (LIP), a truncated form of C/EBP beta, was a potent negative regulator of the promoter. C/EBP alpha also bound to and stimulated the activity of the AFP enhancer at -2.5 kb. Interestingly, HNF-1 beta was found to be more potent than HNF-1 alpha in activating the AFP promoter. This effect was specific, as it did not occur with the rat albumin promoter. HNF-1 beta, which is produced earlier than HNF-1 alpha during liver development, would thus have the greater influence on the AFP promoter in early development. Both HNF-1s allowed expression of the AFP promoter in cells of nonhepatic origin. Overexpression of NF-1 induced a specific decrease in the activity of the AFP promoter. This strongly suggests that competition between NF-1 and HNF-1 for binding to their overlapping binding sites on the AFP promoter is critical for modulating its activity. Thus changing combinations of these trans-acting factors may tightly modulate the AFP promoter activity in the course of liver development and carcinogenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , alfa-Fetoproteínas/genética , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , ADN/genética , Elementos de Facilitación Genéticos , Expresión Génica , Vectores Genéticos , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Datos de Secuencia Molecular , Factores de Transcripción NFI , Regiones Promotoras Genéticas , Ratas , Transfección , Células Tumorales Cultivadas/metabolismo , Proteína 1 de Unión a la Caja YRESUMEN
Metabolic changes during the first 24 hours of starvation were studied in rats previously adapted for 3 weeks during the postweaning growth period to a low-protein diet using lactalbumin as a dietary protein source. Previous adaptation to a high-quality, low-protein diet reduced the effects of early starvation on the loss of body and liver weight. In rats fed a low-protein diet (6% lactalbumin, LP rats), free triiodothyronine (T3) concentration remained higher than in control rats (13% lactalbumin, C rats) throughout the experiment (+38%, 24 hours), and the plasma insulin concentration, which was lower than in C rats during the first 6 hours (-56%), was not different thereafter. Plasma insulin to glucagon molar ratio was lower (-54%) and liver cyclic adenosine monophosphate (cAMP) concentration was higher (+28%) in LP than in C rats in the fed state, but these were not different at 24 hours of starvation. Plasma glucose concentration was slightly lower in LP than in C rats (-15%) in the fed state, but it was not different in both groups during starvation. Whereas they were unchanged in the fed state, plasma lactate concentration was lower (-57%) and free fatty acid and total ketone body concentrations were higher (+38% and +183%, respectively) in LP than in C rats at 24 hours of starvation.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas en la Dieta/administración & dosificación , Glucólisis , Inanición/metabolismo , Animales , Glucemia/análisis , Homeostasis , Hormonas/sangre , Masculino , Fosfoenolpiruvato Carboxiquinasa (GTP)/análisis , Piruvato Quinasa/análisis , Ratas , Ratas Endogámicas , DesteteRESUMEN
Energy intake, weight gain, carcass composition, plasma fuels, hepatic metabolites and lipogenic enzyme activities were studied in adult rats fed either a low fat, high carbohydrate (LF) diet or one of two fat-containing diets in which 32% of the metabolizable energy was constituted by long-chain triglycerides (LCT) or medium-chain triglycerides (MCT). Compared with the LF diet, the MCT diet did not depress food and energy intake, weight gain, energy and nitrogen retention or lipid deposition and did not produce ketogenesis. The weight gain of rats fed LCT was 25% higher, and increased lipid deposition was observed. Lower lipogenic enzyme activities were observed in rats fed the LF diet containing 4% corn oil than in rats fed the MCT diet containing 1% corn oil. This effect disappeared when rats previously adapted to the LCT diet were fed LF or MCT diets containing 1% corn oil for 21 d. By d 21, in both groups, hepatic malic enzyme, ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthase activities were 2.2-, 2.0-, 2.3- and 1.8-fold higher than those of rats fed LCT. Intermediate hepatic glucose-6-phosphate dehydrogenase activities were observed in rats fed the MCT diet, compared with LCT (40% lower) and LF (1.6-fold higher) diets. These data show that in rats fed a diet in which MCT supplies 32% of metabolizable energy, a high activity of lipogenic enzymes is observed, suggesting that MCT had no inhibitory effect on the activity of these enzymes.
Asunto(s)
Carbohidratos de la Dieta/administración & dosificación , Grasas de la Dieta/administración & dosificación , Metabolismo Energético , Hígado/metabolismo , Triglicéridos/administración & dosificación , Animales , Glucemia/análisis , Colesterol/sangre , Ingestión de Energía , Insulina/sangre , Cuerpos Cetónicos/sangre , Lípidos/sangre , Hígado/enzimología , Glucógeno Hepático/análisis , Masculino , Tamaño de los Órganos , Ratas , Aumento de PesoRESUMEN
Food intake, plasma glucose, insulin (I) and glucagon (G), hepatic glycogen and fructose 2,6-bisphosphate (F-2, 6-P2) and liver glucokinase, glucose 6-phosphatase (G6-Pase), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-PF-2 kinase/F-2, 6-P2ase), pyruvate kinase (PK-L) and phosphoenolpyruvate carboxykinase (PEPCK) activities were measured in 2 and 22-month-old rats before 3 d starvation and after 2, 4, 6, 24 and 48 h refeeding a high carbohydrate (HC, 74% w/w) diet. Expressed per 100 g of body weight, the food intake of old rats was 55% lower than that of young rats and the amount of carbohydrate absorbed hourly during the first 6 h of refeeding was 2.4-fold higher in young than in old rats. During the first 6 h of refeeding plasma glucose increased 2-fold and returned to normal values after 24 h in young rats, while plasma glucose did not change during refeeding in old rats. In young rats [I] fell by 85% after starvation and returned to normal values 2 h after refeeding. [I] was higher in old than in young rats; it decreased by 40% after starvation and returned to the basal value 4 h after refeeding. No marked changes were observed in plasma [G] in both groups. No difference was observed in hepatic glycogen in the two groups, while F-2, 6-P2 was higher in old than in young rats. In young rats, the opposite changes in liver glucokinase and G6-Pase activities occurring after starvation and during refeeding were
Asunto(s)
Envejecimiento/metabolismo , Ingestión de Alimentos/fisiología , Gluconeogénesis/fisiología , Glucólisis/fisiología , Hígado/enzimología , Inanición/enzimología , Animales , Carbohidratos de la Dieta/administración & dosificación , Glucagón/sangre , Insulina/sangre , Masculino , Ratas , Ratas EndogámicasRESUMEN
Food intake, plasma glucose, insulin (I) and triiodothyronine (T3) and liver glucose 6-phosphate dehydrogenase (G6P-DH), malic enzyme (ME). ATP-citrate lyase, acetyl-CoA carboxylase (AcCoACx) and fatty acid synthase (FAS) activities were measured in 2 and 22 months old rats before, after 3 d starvation and 2,4,6. 24 and 48 h refeeding a high carbohydrate (74% w/w) diet. Expressed per 100 g of body weight, the carbohydrate intake of old rats was 55% lower than that of young rats. Plasma insulin was higher in old than in young rats and decreased (-40%) after starvation and returned to control values 4 h after refeeding. In young rats plasma insulin fell after starvation (-85%) and returned to normal values 2 h after refeeding. No significant differences were observed in plasma [T3] between the two groups. During the first 6 h of refeeding, plasma glucose increased 2-fold and returned to control values after 24 h in young rats. In old rats, plasma glucose returned to its control value after 2 h. Compared to the starved level, 48 h after refeeding, G6P-DH, ME, ATP-citrate lyase, AcCoACx and FAS activities increased 5- to 6-fold in young rats, while in old rats the increase was much smaller and represented 35% of that observed in young rats. These results suggest, that the age-related reduction in inducibility of hepatic lipogenic enzymes of rats refed a high carbohydrate diet after starvation may be due to a spontaneous decrease in the carbohydrate intake and to a decrease effectiveness of insulin (insulin resistance).
Asunto(s)
Envejecimiento/metabolismo , Alimentos , Lípidos/biosíntesis , Hígado/enzimología , Inanición/enzimología , ATP Citrato (pro-S)-Liasa/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Animales , Glucemia/metabolismo , Ácido Graso Sintasas/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Insulina/sangre , Malato Deshidrogenasa/metabolismo , Masculino , Ratas , Ratas Endogámicas , Triyodotironina/sangreRESUMEN
Metabolic changes in rats fed a low protein diet were investigated during 3 weeks after weaning using lactalbumin (LP) as dietary protein source. The energy intake was higher and the weight gain lower in rats fed the low protein diet (6%, LP group) than in control rats (13% lactalbumin, C group). Low protein diet induced no changes in plasma glucose, free fatty acids, or triacylglycerol concentrations; however, plasma protein and urea concentrations were lower in LP than in C rats. Plasma free T3 was higher in LP than in C rats (+38%, day 21) and insulin progressively decreased during the experimental period (-56%, day 21) without change in glucagon. Liver glycogen and triacylglycerol concentrations (+40% and +180%, respectively, day 21), and cytosolic and mitochondrial redox states increased (+100% and +100%, day 21), and protein concentration was decreased (-15%, day 21). Pyruvate kinase (PK) and malic enzyme activities were higher in LP than in C rats throughout the experiment (+80% and +210%, respectively, day 21), and glucose-6-phosphate dehydrogenase (G6PDH) activity progressively decreased (-65%, day 21). Phosphoenolpyruvate carboxykinase (PEPCK) activity increased after 2 weeks on a LP diet (+35%, day 21) and fatty acid synthetase (FAS) activity increased only during the first week on the diet (+100%, day 7). Such hormonal and metabolic changes appeared to be associated with the development of a futile energy-wasting cycle between pyruvate and phosphoenolpyruvate.
Asunto(s)
Proteínas en la Dieta/administración & dosificación , Crecimiento , Animales , Glucemia/metabolismo , Proteínas Sanguíneas/metabolismo , Ingestión de Energía , Ácidos Grasos no Esterificados/sangre , Glucagón/sangre , Insulina/sangre , Lactalbúmina/administración & dosificación , Hígado/enzimología , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Masculino , Tamaño de los Órganos , Oxidación-Reducción , Ratas , Ratas Endogámicas , Triglicéridos/sangre , Triyodotironina/sangre , Urea/sangre , Destete , Aumento de PesoRESUMEN
This study examined the effect on glycogen resynthesis during recovery from exercise of feeding glucose orally to physically trained rats which had been fed for 5 weeks on high-protein low fat (HP), high-protein/long-chain triglyceride (LCT) or high carbohydrate (CHO) diets. Muscle glycogen remained low and hepatic gluconeogenesis was stimulated by long-term fat or high-protein diets. The trained rats received, via a stomach tube, 3 ml of a 34% glucose solution immediately after exercise (2 h at 20 m.min-1), followed by 1-ml portions at hourly intervals until the end of the experiments. When fed glucose soleus muscle glycogen overcompensation occurred rapidly in the rats fed all three diets following prolonged exercise. In LCT- and CHO-fed rats, glucose feeding appeared more effective for soleus muscle repletion than in HP-fed rats. The liver demonstrated no appreciable glycogen overcompensation. A complete restoration of liver glycogen occurred within a 2- to 4-h recovery period in the rats fed HP-diet, while the liver glycogen store had been restored by only 67% in CHO-fed rats and 84% in LCT-fed rats within a 6-h recovery period. This coincides with low gluconeogenesis efficiency in these animals.
Asunto(s)
Grasas de la Dieta/administración & dosificación , Proteínas en la Dieta/administración & dosificación , Glucosa/administración & dosificación , Glucógeno/biosíntesis , Esfuerzo Físico , Animales , AMP Cíclico/metabolismo , Carbohidratos de la Dieta/administración & dosificación , Carbohidratos de la Dieta/metabolismo , Grasas de la Dieta/metabolismo , Proteínas en la Dieta/metabolismo , Gluconeogénesis , Glucosa/metabolismo , Cinética , Hígado/metabolismo , Glucógeno Hepático/biosíntesis , Masculino , Músculos/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Condicionamiento Físico Animal , Ratas , Ratas EndogámicasRESUMEN
The aim of this work was to find by which mechanisms an increased availability of plasma free fatty acids (FFA) reduced carbohydrate utilization during exercise. Rats were fed high-protein medium-chain triglycerides (MCT), high-protein long-chain triglycerides (LCT), carbohydrate (CHO) or high-protein low-fat (HP) diets for 5 weeks, and liver and muscle glycogen, gluconeogenesis and FFA oxidation were studied in rested and trained runner rats. In the rested state the hepatic glycogen store was decreased by fat and protein feeding, whereas soleus muscle glycogen concentration was only affected by high-protein diets. The percentage decrease in liver and muscle glycogen stores, after running, was similar in fat-fed, high-protein and CHO-fed rats. The fact that plasma glucose did not drastically change during exercise could be explained by a stimulation of hepatic gluconeogenesis: the activity of phosphoenolpyruvate carboxykinase (PEPCK) and liver phosphoenolpyruvate (PEP) concentration increased as well as cyclic adenosine monophosphate (AMPc) while liver fructose 2,6-bisphosphate decreased and plasma FFA rose. In contrast, the stimulation of gluconeogenesis in rested HP-, MCT- and LCT-fed rats appears to be independent of cyclic AMP.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Grasas de la Dieta/administración & dosificación , Proteínas en la Dieta/administración & dosificación , Músculos/metabolismo , Esfuerzo Físico , Animales , Glucemia/análisis , Carbohidratos de la Dieta/administración & dosificación , Ingestión de Alimentos , Ácidos Grasos no Esterificados/sangre , Fructosadifosfatos/metabolismo , Glucógeno/metabolismo , Lactatos/metabolismo , Glucógeno Hepático/metabolismo , Masculino , Fosfoenolpiruvato/metabolismo , Piruvatos/metabolismo , Ratas , Ratas Endogámicas , Factores de Tiempo , Triglicéridos/metabolismo , Aumento de PesoRESUMEN
Plasma insulin (I), glucagon (G) and glucose, hepatic glycogen, fructose 2, 6-bisphosphate (F2, 6-P2), fructose 1, 6-bisphosphate, phosphoenolpyruvate, and some liver key enzymes involved in glycolysis (6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (6-PF-2kinase/F-2,6-P2ase), activity ratio (velocity at suboptimal substrate concentration/maximum velocity) of pyruvate kinase (PK-L] and in gluconeogenesis (phosphoenolpyruvate carboxykinase activity) have been compared in young (2 months) and old (16 months) rats upon starvation or transition to a high protein (HP) diet. In the 10 and 24 hours after the dietary switch, plasma glucose decreased less and hepatic glycogen was less depleted in the old rats. The ratios of plasma I/G and of hepatic 6-PF-2kinase/F-2,6-P2ase were higher in the old rats and their decrease delayed at both time points, as was the concentration of hepatic F-2,6-P2 and the activity ratio of PK-L (before and after removal of endogenous noncovalent factors). The consistency of these differences indicate that the mechanisms for control of glycolysis/gluconeogenesis are similar in young and old rats, but it appears that in old rats starved or fed HP diet, the switch from glycolysis to gluconeogenesis is delayed. This suggests that as a result of the slowness of the hormonal changes the process of phosphorylation/dephosphorylation, which is so important in the short-term regulation of the glycolysis/gluconeogenesis pathway, may be impaired with age.
Asunto(s)
Proteínas en la Dieta/farmacología , Fructosadifosfatos/metabolismo , Hexosadifosfatos/metabolismo , Hígado/crecimiento & desarrollo , Fosfofructoquinasa-1/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Piruvato Quinasa/metabolismo , Inanición , Envejecimiento , Animales , Glucemia/metabolismo , Carbohidratos de la Dieta/farmacología , Cinética , Hígado/enzimología , Glucógeno Hepático/metabolismo , Masculino , Ratas , Ratas Endogámicas , Valores de ReferenciaRESUMEN
The activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), malic enzyme (EC 1.1.1.40), ATP-citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase were lower (-25 to -60%) in liver of rats fed during 45 days with a moderate long-chain triglycerides (LCT) content diet (32% of metabolizable energy, ME), than in control rats fed with a low fat diet (LCT, 10% of ME). However, the fall in malic enzyme activity was not significant. In contrast, these activities were higher (+40 to +160%) in rats fed with a diet with a moderate medium-chain triglycerides (MCT) content (32% of ME), than in control rats. Nevertheless, the increase in activity of malic enzyme and ATP-citrate lyase was more important. Contrary to LCTs, MCTs had no inhibitory effect on the activity of enzymes involved in hepatic lipogenesis.
Asunto(s)
Grasas de la Dieta/farmacología , Hígado/enzimología , Triglicéridos/farmacología , ATP Citrato (pro-S)-Liasa/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Animales , Composición Corporal/efectos de los fármacos , Grasas de la Dieta/administración & dosificación , Ácido Graso Sintasas/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/metabolismo , Masculino , Ratas , Ratas Endogámicas , Triglicéridos/administración & dosificación , Aumento de Peso/efectos de los fármacosRESUMEN
Changes in plasma glucose, hepatic cyclic AMP, glycogen and fructose 2,6-bisphosphate (F-2,6-P2), and liver 6-phosphofructo-2-kinase (6-PF-2kinase), fructose 2,6-bisphosphatase (F-2,6-P2ase) and phosphoenolpyruvate carboxykinase (PEPCK) activities were examined in rats fed a low protein, high carbohydrate (HC) diet during 3 d of either starvation or feeding a high protein, carbohydrate-free (HP) diet. Under both HP feeding or starvation, liver cyclic AMP increased after 1 d and remained constant thereafter. Whereas plasma glucose was low during starvation, it was unaffected by HP feeding. In both experimental groups, liver glycogen fell after 1 d; thereafter it remained low on starvation, but increased progressively on HP diet reaching 70% of the HC-fed rats value on day 3. Under both experimental conditions, F-2,6-P2 fell 85% after day 1 and was unchanged thereafter. One day after the start of starvation or consumption of the HP diet, 6-PF-2kinase decreased, F-2,6-P2ase increased and 6-PF-2kinase/F-2,6-P2ase ratio decreased, but changes were significantly more important with the HP diet than with starvation. PEPCK activity increased in both experimental conditions, but the increase was greater on the HP diet than on starvation. These findings suggest that during the first 3 d the adaptative response of hepatic gluconeogenesis is higher with a HP diet than upon starvation.
Asunto(s)
Proteínas en la Dieta , Fructosadifosfatos/metabolismo , Hexosadifosfatos/metabolismo , Hígado/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Glucemia/metabolismo , AMP Cíclico/metabolismo , Cinética , Glucógeno Hepático/metabolismo , Masculino , Fosfofructoquinasa-2 , Ratas , Ratas Endogámicas , Valores de Referencia , InaniciónRESUMEN
Energy intake, weight gain, carcass composition, plasma hormones and fuels, hepatic metabolites and the activities of phosphoenolpyruvate carboxykinase (PEPCK), malic enzyme, and glucose 6-phosphate dehydrogenase (G6P-DH) were examined in adult rats during a 44-day period of low fat, high carbohydrate (LF) feeding or of consumption of one or two high (70% metabolizable energy) fat diets composed of 63% (metabolizable energy) long-chain (LCT) or medium-chain (MCT) triglycerides. Energy intake was similar in the LCT and MCT groups but was less than that of LF group. The weight gain of rats fed MCT diet was 30% less than that of rats fed LF or LCT diets. Energy retention was less when the diet provided MCT than LCT or LF, and that resulted in a 60% decrease in the daily lipids deposition. Plasma glucose, free fatty acids, glycerol, and insulin/glucagon ratio were similar in the three groups. Blood ketone body (KB) concentrations in rats fed the high fat diets were extremely elevated, particularly in the MCT group, but declined throughout the experiment and by the 44th day hyperketonemia decreased by 50% but remained higher than in the LF diet. The blood beta-hydroxybutyrate/acetoacetate (B/A) ratio remained slightly elevated in rats fed the high fat diets. Similar changes were observed in liver KB concentration and in the B/A ratio. Liver lactate/pyruvate ratio elevated in the LCT and MCT groups at the initiation of the diets decreased by 50% at the end of the experiment. The consumption of high fat diets led to a 1.5-fold increase in liver PEPCK activity.(ABSTRACT TRUNCATED AT 250 WORDS)