9 research outputs found
Regulation by insulin of brush border membrane hydrolases in the rat small intestine
Les travaux antérieurs du laboratoire ont démontré que l'administration d'insuline à des ratons non-sevrés induisait de manière prématurée la croissance intestinale et l'activité enzymatique au niveau de la membrane de la bordure en brosse (BBM). Le(s) mécanisme(s) par le(s)quel(s) l'insuline stimule l'activité des hydrolases demeure(nt) inconnu(s). L'objectif de notre travail était d'étudier ces mécanismes. Nous avons donc réalisé les expériences suivantes: primo, nous avons analysé la distribution, l'ontogenèse et les propriétés moléculaires des récepteurs à l'insuline (IR), ainsi que ses substrats dans les entérocytes matures et immatures du rat ; secundo, nous avons comparé les réponses de quatre enzymes de la BBM (SI, maltase, LPH, et aminopeptidase) après l'administration exogène d'insuline, de l'analogue Asp-B10, de l'IGF-I et d'un anticorps monoclonal anti-récepteur à l'insuline [MAb (IR)]; tertio, nous avons déterminé l'activité de la PKB (reflètant l'activation de la voie de PI 3-kinase) et des p42/p44 MAP kinase, deux voies de signalisation importantes, qui sont activées par une stimulation des entérocytes à l'insuline et qui peuvent être impliquées dans l'induction prématurée des hydrolases de BBM; quatro, nous avons déterminé si les p42/p44 et la p38 MAP kinase pouvaient jouer un rôle dans la transmission du signal de l'insuline, qui stimule les hydrolases microvillositaires. Nos résultats suggèrent que l'induction prématurée de la SI et la stimulation de la maltase par l'insuline, sont déterminées par un signal dose-dépendant déclenché par la liaison de l'insuline à son récepteur intestinal activant ERKs. ERKs phosphorylent ainsi un ou plusieurs facteur(s) de transcription(s) stimulant la transcription de gene de la SI.Our previous studies have shown that administration of insulin to suckling rats induced premature intestinal growth and the expression of brush border membrane (BBM) enzymes. The mechanism(s) by which insulin prematurely enhances the activity of BBM hydrolases in the immature rat small intestine is unknown. The objective of our study was to investigate these mechanisms. Therefore, the following experiments were conducted: first- we have analyzed the distribution, ontogeny and molecular properties of IRs and the receptor-related substrates in enterocytes from mature and immature rats; second- we have compared the responses of four BBM enzymes (SI, maltase, LPH and aminopeptidases) to exogenous insulin, to the analog B-Asp10, to IGF-I and to MAb anti-IR monoclonal given to preweaned rats; third- we have determined the activity of PKB (reflecting the activation of PI 3-kinase pathway) and p42/p44 MAP kinase, two important postreceptor signaling pathways that were stimulated by insulin and could be involved in the transmission of the insulin signal which prematurely enhanced BBM hydrolases in the small intestine of rats; fourth- we have determined whether p42/p44 MAP kinases and p38 MAP kinase could be involved in the signal transduction of insulin that stimulates BBM hydrolases in enterocytes from mature and immature rats. Our results suggest that the premature induction of SI by insulin is mediated by one or more transcription factor (s) that would be phosphorylated by activated ERKs and stimulate the SI gene transcription.Thèse de doctorat en sciences biomédicales (SBIM 3) -- UCL, 200
Short-term effects of a physical activity intervention on obesity and aerobic fitness of adolescent girls
Background: In the past two decades, physical activity has decreased during both childhood and adolescence, and particularly adolescence. It seems that schools are attractive settings in which to implement interventions designed in order to promote physical activity in children; but in Iranian students, few studies have evaluated the effects of such interventions on overweight and obese children. The aim of this study was to evaluate the effects of a short-term school-based physical activity on obesity and aerobic fitness in 12-14 years aged girls.
Methods: This is a study with single group pretest and posttest design, in which 129 middle school girls in city of Isfahan were assessed based on preventive plan of inactivity in children at the Provincial Health Office. Variables, including weight, height, body mass index (BMI), waist-hip ratio (WHR), body fat percentage and aerobic power of subjects were measured using valid tests.
Results: This study showed that subjects′ body fat percentage changed about 3.6% (37.74% pretest vs. 36.39% posttest), VO 2 max changed 7.43% (29.72 pretest vs. 31.93 posttest), WHR changed 1.12% (0.89 pretest vs. 0.88 posttest), whereas BMI was changed 1.65% (27.80 pretest vs. 27.34 posttest). Findings also revealed that there were significant differences between fat percent, (P = 0.001) and VO 2 max (P = 0.001) of subjects, but there was no difference between BMI of them in pre- and post-tests (P = 0.361).
Conclusions: These results suggest that even a short-term exercise intervention may lead to positive changes in body fat percentage, WHR and aerobic fitness of overweight children. Therefore, school-based physical activity interventions can be an effective preventive strategy to control obesity and overweight in students
Expression of insulin receptors and of 60-kDa receptor substrate in rat mature and immature enterocytes
The mechanism(s) by which rat immature enterocytes exhibit increased responsiveness to insulin before weaning is unknown. Therefore, we have analyzed the distribution, ontogeny, and molecular properties of insulin receptors (IR) and of related substrates in immature and mature enterocytes. IR were studied by radioligand binding assays, cross-linking labeling, immunohistochemistry, and in vitro phosphorylated substrates by immunoprecipitation. Regardless of age, 125I-insulin binding to IR was five times higher in crypt cells than in villus cells and two times higher in the ileum than in the jejunum. Binding capacity to villus cells from sucklings (day 14) exceeded three times that of older animals (day 30 and day 60). Scatchard analysis of equilibrium binding data confirmed an age-related decrease in low- and high-affinity receptor classes without change in affinity constants. In concordance, both alpha- and beta-IR subunits were more abundant in immature than in mature membranes. In vitro, insulin elicited the phosphorylation of three membrane proteins (96, 60 and 42 kDa), whose signals were virtually inhibited by preincubating membranes with antireceptor monoclonal antibodies. By immunoprecipitation, the 60-kDa signal was rapidly detected as a tyrosine-phosphorylated protein, expressed in mature and immature membranes, and identified as a receptor substrate phosphorylated in vitro by the IR tyrosine kinase. In conclusion, 1) increased responsiveness of rat immature enterocytes to insulin could be related to high membrane concentrations of IR and 2) normal rat enterocytes express a 60-kDa phosphotyrosine protein identified as a direct substrate of the IR tyrosine kinase
Insulin signal transduction in rat small intestine: role of MAP kinases in expression of mucosal hydrolases
The postreceptor events regulating the signal of insulin downstream in rat intestinal cells have not yet been analyzed. Our objectives were to identify the nature of receptor substrates and phosphorylated proteins involved in the signaling of insulin and to investigate the mechanism(s) by which insulin enhances intestinal hydrolases. In response to insulin, the following proteins were rapidly phosphorylated on tyrosine residues: 1) insulin receptor substrates-1 (IRS-1), -2, and -4; 2) phospholipase C-isoenzyme-gamma; 3) the Ras-GTPase-activating protein (GAP) associated with Rho GAP and p62(Src); 4) the insulin receptor beta-subunit; 5) the p85 subunits of phosphatidylinositol 3-kinase (PI 3-kinase); 6) the Src homology 2 alpha-collagen protein; 7) protein kinase B; 8) mitogen-activated protein (MAP) kinase-1 and -2; and 9) growth receptor-bound protein-2. Compared with controls, insulin enhanced the intestinal activity of MAP kinase-2 and protein kinase B by two- and fivefold, respectively, but did not enhance p70/S6 ribosomal kinase. Administration of an antireceptor antibody or MAP-kinase inhibitor PD-98059 but not a PI 3-kinase inhibitor (wortmannin) to sucklings inhibited the effects of insulin on mucosal mass and enzyme expression. We conclude that normal rat enterocytes express all of the receptor substrates and mediators involved in different insulin signaling pathways and that receptor binding initiates a signal enhancing brush-border membrane hydrolase, which appears to be regulated by the cascade of MAP kinases but not by PI 3-kinase
Ontogeny of MAP kinases in rat small intestine: premature stimulation by insulin of BBM hydrolases is regulated by ERKs but not by p-38 MAP kinase.
Although mitogen-activating protein (MAP) kinases are crucial signal transduction molecules regulating cellular proliferation, differentiation, and morphology, their ontogenic changes in the small intestine have not been analyzed. Also, it remains unknown which pathway of activated MAP kinases regulates the expression of brush border membrane hydrolases during growth. Therefore, we have analyzed the mucosal distribution, ontogeny, and responses to insulin and to inhibitors of p44, p42, and p38 MAP kinases in immature and mature enterocytes using Western blot analysis and autoradiography after immunoprecipitation, immunohistochemistry, and in vitro phosphorylation assays. Between d 10 and 40 postpartum, diphosphorylated active p44/p42 extracellular regulated protein kinases (ERKs) increased in abundance compared with total immunoprecipitated ERKs, and were highly responsive to exogenous insulin. In concordance, ERK total activity increased by 4-fold during the same period of growth and was further enhanced 2-fold by exogenous insulin. In weaning rats, ERKs were mainly located in membranes of villus cells and with less intensity in crypt cells. By contrast, p38 MAP kinase was unresponsive to insulin and was confined to nuclei. Administration to sucklings of PD 098059, a specific inhibitor of ERKs, not only inhibited the premature stimulation of sucrase, lactase, and maltase total activities in response to exogenous insulin, but also depressed the natural expression of these brush border membrane enzymes in the absence of insulin stimulation. In concordance, administration of SB 203580, a specific inhibitor of p38 MAP kinase, failed to inhibit both the response of brush border membrane hydrolases to insulin and their natural expression in the absence of insulin stimulation. We conclude that the ontogenic expression of brush border membrane hydrolases and their premature stimulation by insulin are regulated at least in part by the activation of p44/p42 ERKs but not by p38 MAP kinase
Ontogeny of MAP Kinases in Rat Small Intestine: Premature Stimulation by Insulin of BBM Hydrolases Is Regulated by ERKs but not by p-38 MAP Kinase
Saccharomyces boulardii enhances N-terminal peptide hydrolysis in suckling rat small intestine by endoluminal release of a zinc-binding metalloprotease.
Saccharomyces boulardii (S. boulardii), a biotherapeutic agent effective in acute and chronic enterocolopathies, produces trophic intestinal effects at least in part mediated by the endoluminal release of polyamines. However, the effects of the yeast on peptide hydrolysis have not yet been studied. The objectives of this study were to assess in suckling rats the endoluminal and mucosal aminopeptidase activities in response to S. boulardii treatment and to analyze their related mechanisms. Peptidase activities were assayed on yeast cells by using several L-amino acid-p-nitroanilide substrates in the pH range of 2 to 10. A marked hydrolytic activity was found for L-leucine-p-nitroanilide that peaked at pH = 8 (K(m) = 0.334 mM, V(max) = 44.7 micromol.min(-1).g(-1) protein). N-terminal peptide hydrolysis was confirmed using as substrate L-Leu-Gly-Gly (K(m) = 4.71 mM, V(max) = 18.08 micromol.min(-1).g(-1) protein). Enzyme reactions were inhibited in the presence of 1 mM Zn(2+). Oral treatment of sucklings with S. boulardii significantly enhanced jejunal and ileal mucosal leucine-aminopeptidase activities by 24 and 34%, respectively, over controls. In concordance, aminopeptidase activity was enhanced in jejunal and ileal endoluminal fluid samples by 47 and 105%, respectively. By use of an IgG-purified antibody raised against the zinc-binding domain common to metalloproteases, the yeast aminopeptidase was immunoprecipitated and detected as an heteromeric enzyme of 108 and 87-kD subunits. S. boulardii, when given orally to suckling rats, is able to significantly enhance hydrolysis of N-terminal oligopeptides in both endoluminal fluid and intestinal mucosa by the endoluminal release of a leucine aminopeptidase that appears to be a zinc-binding metalloprotease belonging to the M1 family of peptidases