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Top of pageAbstractDiabetic pregnancy results in several metabolic and hormonal disorders cartier pink gold love bracelet, both in the embryo and the fetus of different species, including humans. Insulin is a potent modulator of brain development and is suggested to promote the differentiation and maturation of hypothalamic or related extrahypothalamic structures, which are directly involved in neural inputs to the pancreas. Because these structures are known to be specifically responsive both to insulin and glucose, we examined the effects of 48 h hyperglycemic clamps in unrestrained pregnant rats on insulin binding and glucose transporter expression in hypothalamic and extrahypothalamic related areas of their fetal offspring. The deleterious effect of brain hyperinsulinism during the late gestational stage does not seem to act through glucose transporter (GLUT) expression, inasmuch as no relationship between GLUT level and hyperinsulinism in brain areas could be observed. This situation seems to be mainly related to intrauterine hyperglycemia, inasmuch as mild hyperglycemia induced by glucose infusion during late gestation in normal rats is sufficient to induce persistent impairment of glucose regulation and insulin secretion in the adult offspring born of hyperglycemic mothers (4, 5). Interestingly, in this model, insulin deficiency was due to a perturbation of the control of insulin secretion by the ANS and not to an intrinsic pancreatic defect (5). The activity of the ANS is under the influence of hypothalamic nuclei (PVN, VMH, and LH), themselves connected to preganglionic nuclei in the brainstem (6, 7). These areas present a neuronal population able to respond electrophysiologically to insulin and glucose (8, 9). Insulin, insulin receptors, and glucose transporters are present in the brain (8, 10). The defect in insulin secretion observed in the offspring of hyperglycemic mothers could be linked to impaired glucose and/or insulin sensing in the brain. We hypothesized that fetal hyperglycemia and/or hyperinsulinemia could hamper the functional development of glucose and/or insulin sensitive brain areas. In fact, previous studies in models of perinatal hyperinsulinism (where hypothalamic insulin binding was increased) have demonstrated that hypothalamic nuclei were structurally altered in adult life (11, 12). We first addressed the question of whether hyperglycemia and/or hyperinsulinemia during late pregnancy alter insulin binding in fetal brain areas involved in the autonomic control of insulin secretion and glucose homeostasis.Insulin controls glucose transport in insulin sensitive tissues through GLUT4 (10). Some studies have reported that, in the brain, insulin stimulates glucose transport in vitro in glial cells (13). In vivo, hyperglycemia and hyperinsulinemia lead to increased glucose utilization in the hypothalamus (14). Altogether, these data suggest that insulin might be involved in glucose transport in some specific brain areas. Among the neural GLUT identified, GLUT1 (endothelial, glial cells) and GLUT3 (neuronal cells) are the main isoforms (10). We have recently demonstrated that GLUT2 and GLUT4 were also, at lower levels, expressed in adult hypothalamic and spinal cord areas, respectively, in astrocytic and neuronal cells (15 replica cartier bracelet, 16). Modifying the GLUT2 expression in the brain led to an abnormal control of energy metabolism and deranged insulin secretion (17). Moreover, in physiopathological model such as Z rats, GLUT4 expression was altered in hypothalamic nuclei (18).In the second part of the study, we focused on the fact that a possible consequence of an alteration in insulin binding to specific brain areas could result in the modifications of expression of isoforms GLUT1, GLUT2, GLUT3, and GLUT4 in these areas during late development in the rat fetus.The study was performed on rat fetuses at term from mothers made mildly hyperglycemic during the last 2 d of pregnancy by continuous glucose infusion. Three month old pregnant female Wistar rats weighing 250 g were used. They were allowed free access to water and standard laboratory chow pellets (UAR 113, Usine d'Alimentation Rationnelle, Villemoisson, France). At d 19 of pregnancy, a catheter was implanted under intraperitoneal pentobarbital anesthesia (125 mg/kg) in the right atrium via the jugular vein. The technique (19) for long term infusion in unrestrained rats was used for glucose infusion (hypertonic 30 sterile glucose, Chaix Du Marais, Paris, France). The infusion period started on d 2 after surgery and lasted 2 d. The initial infusion rate was 30 Control rats were infused with 0.9 NaCl. Blood glucose of infused rats was measured twice daily in samples drawn from caudal vessels. These controls allowed us to adjust glucose flow rate throughout the infusion to maintain mild hyperglycemia. After centrifugation, the remaining plasma was stored at until insulin assay. Fetal blood samples originating from axillary vessels were used for measurements of both glycemia and insulinemia. Plasma immunoreactive insulin was measured by a RIA kit (DiaSorin, Saluggia, Italy).At 21.5 d of pregnancy (normal term, 22 d), the infusions were disrupted and the fetuses were immediately removed after cesarean section. They were successively exteriorized from the uterus, leaving placenta and umbilical cord in situ; blood samples were taken from the axillary vessels, and the brains were rapidly removed and placed into freezing isopentane ( for 40 s. Fetal brains were stored until cryosection (20). Coronal sections (300 were obtained using a cryostat and placed upon chilled microscope slides. Whole hypothalamus and rostral spinal cord were microdissected and used for either Northern blot, qcRT PCR, or Western blot. Autoradiograms were obtained by apposition of the sections on tritiated ultrafilm (LKB ultrafilm, Amersham Biosciences AB, Uppsala, Sweden) over 7 d. Six to 10 readings were made for each discrete structure in each animal. Brain mRNA was further purified using an mRNA extraction kit (Amersham Biosciences AB) and the concentrations measured by absorbance at 260 nm. A Northern blot was performed, as described (22), using 20 of purified mRNA. The blots were hybridized with 32P labeled probe using a Megaprime labeling system kit (Amersham Biosciences AB). The probes for GLUT1, GLUT3, and GLUT4 corresponded to the 541 597 and 334 cDNA sequences, respectively, and were constructed using PCR. Blots were exposed at with intensifying screens. hypothalamus and rostral spinal cord). Samples were stored at until assays. Fifty microgram membrane samples were analyzed for GLUT content by classical Western blot analysis with the rabbit polyclonal GLUT1 (1/15,000) antibody (purchased from Biogenesis, Argene, France) and the goat polyclonal GLUT3 and GLUT4 antibodies (Santa Cruz Biotechnology, Tebu, France), 1/5000 and 1/4500, respectively. The immunoblot signals were detected using the Amersham ECL detection kit system. Films of GLUT1, GLUT3, GLUT4, and were quantified as described for Northern blots. Results were normalized by quantification of corresponding levels of protein. Statistical significance was accepted at p 0.05. For plasma insulin (Table 1), a 5 fold increase was observed in glucose infused mothers and a nearly 7 fold increase was observed in their fetuses, compared with controls. The greatest differences with controls were observed in the VMH, the AN, and the LH for hypothalamic areas, and in the NTS for extrahypothalamic areas (Fig. 1). PCX, parietal cortex; FCX, frontal cortex; OB, olfactory bulbs; VMH, ventromedial hypothalamus; AN, arcuate nucleus; LH, lateral hypothalamus; AB, amygdaloid body; NTS, nucleus of the tractus solitarius; Ca3, field CA3 of Ammon's horn (hippocampus). Values are the mean SEM. classic Northern blot using mRNA preparation or the highly sensitive qcRT PCR method (data not shown).As shown in Figure 2, exposure of fetuses to hyperglycemia and hyperinsulinemia resulted in a marked increase in GLUT1 and GLUT3 mRNA concentrations, and a less pronounced increase in GLUT4 mRNA at the level of the rostral spinal cord (which includes the NTS). In contrast, hypothalamic GLUT3 and GLUT4 mRNA expression was not altered in hyperglycemic fetuses cartier love bracelet knock off. Only a slight increase of GLUT1 transcripts could be observed (Fig. 2). Five samples per region from 9 to 12 animals per group were analyzed. Results were normalized by quantitation of corresponding mRNA levels. Hypot, hypothalamus; SpCord, rostral spinal cord. Values are the mean SEM. 3A). The quantification of GLUT1, GLUT3, and GLUT4 levels in these samples, expressed as arbitrary standard units relative to the control offspring, is presented in Figure 3B. Hypothalamic as well as rostral spinal cord GLUT3 and GLUT4 levels were unchanged in HG HI fetuses when compared with controls. The 55 kD GLUT1 (endothelial isoform) was also unaffected by the treatment, whatever the area studied. The 45 kD GLUT1 (glial and neuronal isoform) showed a significant increase, but only in the rostral spinal cord; there was no alteration in the hypothalamus.Figure 3.(A) Photographs of GLUT1, GLUT3, and GLUT4 Western blots. Only for the GLUT4 Western blot is presented. Similar images of have been obtained for the other GLUT Western blots. For details, see B and and Methods. (B) GLUT1, GLUT3, and GLUT4 proteins in hypothalamic and in rostral spinal cord brain membranes from control and HI HG fetuses. Three samples per region from 8 to 12 animals per group were analyzed. A 50 membrane fraction was analyzed for each GLUT content (for details, see and Methods Values are mean SEM. 0.05 in hyperglycemic compared with control animals.
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