Placental glucose transporter 3 (GLUT3) is up-regulated in human pregnancies complicated by late-onset intrauterine growth restriction.

INTRODUCTION: Transport of glucose from maternal blood across the placental trophoblastic tissue barrier is critical to sustain fetal growth. The mechanism by which GLUTs are regulated in trophoblasts in response to ischemic hypoxia encountered with intrauterine growth restriction (IUGR) has not been suitably investigated. OBJECTIVE: To investigate placental expression of GLUT1, GLUT3 and GLUT4 and possible mechanisms of GLUT regulation in idiopathic IUGR. METHODS: We analyzed clinical, biochemical and histological data from placentas collected from women affected by idiopathic full-term IUGR (n = 10) and gestational age-matched healthy controls (n = 10). RESULTS: We found increased GLUT3 protein expression in the trophoblast (cytotrophoblast greater than syncytiotrophoblast) on the maternal aspect of the placenta in IUGR compared to normal placenta, but no differences in GLUT1 or GLUT4 were found. No differential methylation of the GLUT3 promoter between normal and IUGR placentas was observed. Increased GLUT3 expression was associated with an increased nuclear concentration of HIF-1α, suggesting hypoxia may play a role in the up-regulation of GLUT3. DISCUSSION: Further studies are needed to elucidate whether increased GLUT3 expression in IUGR is a marker for defective villous maturation or an adaptive response of the trophoblast in response to chronic hypoxia. CONCLUSIONS: Patients with IUGR have increased trophoblast expression of GLUT3, as found under the low-oxygen conditions of the first trimester.

Neuronal glucose transporter isoform 3 deficient mice demonstrate features of autism spectrum disorders.

Neuronal glucose transporter (GLUT) isoform 3 deficiency in null heterozygous mice led to abnormal spatial learning and working memory but normal acquisition and retrieval during contextual conditioning, abnormal cognitive flexibility with intact gross motor ability, electroencephalographic seizures, perturbed social behavior with reduced vocalization and stereotypies at low frequency. This phenotypic expression is unique as it combines the neurobehavioral with the epileptiform characteristics of autism spectrum disorders. This clinical presentation occurred despite metabolic adaptations consisting of an increase in microvascular/glial GLUT1, neuronal GLUT8 and monocarboxylate transporter isoform 2 concentrations, with minimal to no change in brain glucose uptake but an increase in lactate uptake. Neuron-specific glucose deficiency has a negative impact on neurodevelopment interfering with functional competence. This is the first description of GLUT3 deficiency that forms a possible novel genetic mechanism for pervasive developmental disorders, such as the neuropsychiatric autism spectrum disorders, requiring further investigation in humans.

Glucose transporter protein responses to selective hyperglycemia or hyperinsulinemia in fetal sheep.

The acute effect of selective hyperglycemia or hyperinsulinemia on late gestation fetal ovine glucose transporter protein (GLUT-1, GLUT-3, and GLUT-4) concentrations was examined in insulin-insensitive (brain and liver) and insulin-sensitive (myocardium and fat) tissues at 1, 2.5, and 24 h. Hyperglycemia with euinsulinemia caused a two- to threefold increase in brain GLUT-3, liver GLUT-1, and myocardial GLUT-1 concentrations only at 1 h. There was no change in GLUT-4 protein amounts at any time during the selective hyperglycemia. In contrast, selective hyperinsulinemia with euglycemia led to an immediate and persistent twofold increase in liver GLUT-1, which lasted from 1 until 24 h with a concomitant decline in myocardial tissue GLUT-4 amounts, reaching statistical significance at 24 h. No other significant change in response to hyperinsulinemia was noted in any of the other isoforms in any of the other tissues. Simultaneous assessment of total fetal glucose utilization rate (GURf) during selective hyperglycemia demonstrated a transient 40% increase at 1 and 2.5 h, corresponding temporally with a transient increase in brain GLUT-3 and liver and myocardial GLUT-1 protein amounts. In contrast, selective hyperinsulinemia led to a sustained increase in GURf, corresponding temporally with the persistent increase in hepatic GLUT-1 concentrations. We conclude that excess substrate acutely increases GURf associated with an increase in various tissues of the transporter isoforms GLUT-1 and GLUT-3 that mediate fetal basal glucose transport without an effect on the GLUT-4 isoform that mediates insulin action. This contrasts with the tissue-specific effects of selective hyperinsulinemia with a sustained increase in GURf associated with a sustained increase in hepatic basal glucose transporter (GLUT-1) amounts and a myocardial-specific emergence of mild insulin resistance associated with a downregulation of GLUT-4.

Effects of selective hyperglycemia and hyperinsulinemia on glucose transporters in fetal ovine skeletal muscle.

We measured net fetal glucose uptake rate from the placenta, shown previously to be equal to total fetal glucose utilization rate (GUR(f)) and proportional to fetal hindlimb skeletal muscle glucose utilization, under normal conditions and after 1, 2.5, and 24 h of selective hyperglycemia increasing G or selective hyperinsulinemia increasing I. We simultaneously measured the amount of Glut 1 and Glut 4 glucose transporter proteins in fetal sheep skeletal muscle. With increasing G , GUR(f) was increased approximately 40% at 1 and 2.5 h but returned to the control rate by 24 h. This transient increasing G -specific increasing GUR(f) was associated with increased plasma membrane-associated Glut 1 (4-fold) and intracellular Glut 4 (3-fold) protein beginning at 1 h. With increasing I, GUR(f) was increased approximately 70% at 1, 2.5, and 24 h. This more sustained increasing I-specific increasing GUR(f) was associated with a significant increase in Glut 4 protein (2-fold) at 2.5 h but no change in Glut 1 protein. These results show that increasing G and increasing I have independent effects on the amount of Glut 1 and Glut 4 glucose transporter proteins in ovine fetal skeletal muscle. These effects are time dependent and isoform specific and may contribute to increased glucose utilization in fetal skeletal muscle. The lack of a sustained temporal correlation between the increase in transporter proteins and glucose utilization rates indicates that subcellular localization and activity of a transporter or tissues other than the skeletal muscle contribute to net GUR(f).

Myocardial gene expression of glucose transporter 1 and glucose transporter 4 in response to uteroplacental insufficiency in the rat.

Uteroplacental insufficiency causes intrauterine growth retardation (IUGR) and subsequent low birth weight, which predisposes the affected newborn towards adult Syndrome X. Individuals with Syndrome X suffer increased morbidity from adult ischemic heart disease. Myocardial ischemia initiates a defensive increase in cardiac glucose metabolism, and individuals with Syndrome X demonstrate reduced insulin sensitivity and reduced glucose uptake. Glucose transporters GLUT1 and GLUT4 facilitate glucose uptake across cardiac plasma membranes, and hexokinase II (HKII) is the predominant hexokinase isoform in adult cardiac tissue. We therefore hypothesized that GLUT1, GLUT4 and HKII gene expression would be reduced in heart muscle of growth-retarded rats, and that reduced gene expression would result in reduced myocardial glucose uptake. To prove this hypothesis, we measured cardiac GLUT1 and GLUT4 mRNA and protein in control IUGR rat hearts at day 21 and at day 120 of life. HKII mRNA quantification and 2-deoxyglucose-uptake studies were performed in day-120 control and IUGR cardiac muscle. Both GLUT1 and GLUT4 mRNA and protein were significantly reduced at day 21 and at day 120 of life in IUGR hearts. HKII mRNA was also reduced at day 120. Similarly, both basal and insulin-stimulated glucose uptake were significantly reduced in day-120 IUGR cardiac muscle. We conclude that adult rats showing IUGR as a result of uteroplacental insufficiency express significantly less cardiac GLUT1 and GLUT4 mRNA and protein than control animals (which underwent sham operations), and that this decrease in gene expression occurs in parallel with reduced myocardial glucose uptake. We speculate that this reduced GLUT gene expression and glucose uptake contribute towards mortality from ischemic heart disease seen in adults born with IUGR.

Demonstration of direct effects of growth hormone on neonatal cardiomyocytes.

The cellular and molecular basis of growth hormone (GH) actions on the heart remain poorly defined, and it is unclear whether GH effects on the myocardium are direct or mediated at least in part via insulin-like growth factor (IGF-1). Here, we demonstrate that the cultured neonatal cardiomyocyte is not an appropriate model to study the effects of GH because of artifactual loss of GH receptors (GHRs). To circumvent this problem, rat neonatal cardiomyocytes were infected with a recombinant adenovirus expressing the murine GHR. Functional integrity of GHR was suggested by GH-induced activation of the cognate JAK2/STAT5, MAPK, and Akt intracellular pathways in the cells expressing GHR. Although exposure to GH resulted in a significant increase in the size of the cardiomyocyte and increased expression of c-fos, myosin light chain 2, and skeletal alpha-actin mRNAs, there were no significant changes in IGF-1 or atrial natriuretic factor mRNA levels in response to GH stimulation. In this model, GH increased incorporation of leucine, uptake of palmitic acid, and abundance of fatty acid transport protein mRNA. In contrast, GH decreased uptake of 2-deoxy-d-glucose and levels of Glut1 protein. Thus, in isolated rat neonatal cardiomyocytes expressing GHR, GH induces hypertrophy and causes alterations in cellular metabolic profile in the absence of demonstrable changes in IGF-1 mRNA, suggesting that these effects may be independent of IGF-1.

Uteroplacental insufficiency lowers the threshold towards hypoxia-induced cerebral apoptosis in growth-retarded fetal rats.

Infants suffering uteroplacental insufficiency and hypoxic ischemic injury often demonstrate cerebral apoptosis. Our objective was to determine the global effects of uteroplacental insufficiency upon cerebral gene expression of the apoptosis related proteins Bcl-2 and Bax and their role in increasing vulnerability to hypoxia-induced cerebral apoptosis. We therefore caused uteroplacental insufficiency and growth retardation by performing bilateral uterine artery ligation upon pregnant rats 2 days prior to term delivery and elicited further perinatal fetal hypoxia by placing maternal rats in 14% FiO(2) 3 h prior to delivery. We quantified cerebral levels of Bcl-2 and Bax mRNA, lipid peroxidation, caspase-3 activity, and cAMP in control and growth retarded term rat pups that experienced either normoxia or hypoxia. Uteroplacental insufficiency alone caused a significant decrease in cerebral Bcl-2 mRNA levels without altering cerebral Bax mRNA levels, malondialdehyde levels, or caspase-3 activity. In contrast, uteroplacental insufficiency and subsequent fetal hypoxia significantly increased cerebral Bax mRNA levels, lipid peroxidation and caspase-3 activity; Bcl-2 mRNA levels continued to be decreased. Hypoxia alone increased cerebral cAMP levels, whereas uteroplacental insufficiency and subsequent hypoxia decreased cerebral cAMP levels. We speculate that the decrease in Bcl-2 gene expression increases the vulnerability towards cerebral apoptosis in fetal rats exposed initially to uteroplacental insufficiency and subsequent hypoxic stress.

Uteroplacental insufficiency alters hepatic fatty acid-metabolizing enzymes in juvenile and adult rats.

Multiple adult morbidities are associated with intrauterine growth retardation (IUGR) including dyslipidemia. We hypothesized that uteroplacental insufficiency and subsequent IUGR in the rat would lead to altered hepatic fatty acid metabolism. To test this hypothesis, we quantified hepatic mRNA levels of acetyl-CoA carboxylase (ACC), carnitine palmitoyltransferase (CPTI), the beta-oxidation-trifunctional protein (HADH), fasting serum triglycerides, and hepatic malonyl-CoA levels at different ages in control and IUGR rats. Fetal gene expression of all three enzymes was decreased. Juvenile gene expression of CPTI and HADH continued to be decreased, whereas gene expression of ACC was increased. Serum triglycerides were unchanged. A sex-specific response was noted in the adult rats. In males, serum triglycerides, hepatic malonyl-CoA levels, and ACC mRNA levels were significantly increased, and CPTI and HADH mRNA levels were significantly decreased. In contrast, the female rats demonstrated no significant changes in these variables. These results suggest that uteroplacental insufficiency leads to altered hepatic fatty acid metabolism that may contribute to the adult dyslipidemia associated with low birth weight.

Neurohumoral regulation of body weight gain.

The regulation of body weight is a complex process which relies on a balance between supply of nutrients and demand on these nutrients in the form of energy expenditure. Various central and peripheral mechanisms play a crucial role in maintaining this balance. While various neuropeptides in the central nervous system (CNS), particularly in the hypothalamus, maintain the necessary harmony between hyperphagia and anorexia, peripheral signals arising from the gastrointestinal tract (cholecystokinin-8 [CCK-8], amylin), pancreas (insulin) and adipose tissue (leptin) provide the necessary stimuli or a feedback inhibition for the synthesis and secretion of these hypothalamic neuropeptides. Various metabolites of the carbohydrate and fat metabolism are also involved in regulating the neuronal activity in the hypothalamus which ultimately leads to a release of key neuropeptides. In addition to the central mechanisms, peripheral mechanisms that regulate energy expenditure, particularly in the brown adipose tissue and skeletal muscle, are critical in maintaining the overall balance. Insight into these mechanisms sets the stage for developing novel strategies in the treatment of emerging childhood diseases such as obesity, anorexia nervosa, and bulimia. Further, delineation of these processes in the fetus and newborn sets the stage for investigating their role in molding the adult phenotype due to intrauterine adaptations.

Time-dependent physiological regulation of ovine placental GLUT-3 glucose transporter protein.

We immunolocalized the GLUT-3 glucose transporter isoform versus GLUT-1 in the late-gestation epitheliochorial ovine placenta, and we examined the effect of chronic maternal hyperglycemia and hypoglycemia on placental GLUT-3 concentrations. GLUT-3 was limited to the apical surface of the trophoectoderm, whereas GLUT-1 was on the basolateral and apical surfaces of this cell layer and in the epithelial cells lining the placental uterine glands. GLUT-3 concentrations declined at 17-20 days of chronic hyperglycemia (P < 0.05), associated with increased uterine and uteroplacental net glucose uptake rate, but a normal fetal glucose uptake rate was observed. Chronic hypoglycemia did not change GLUT-3 concentrations, although uterine, uteroplacental, and fetal net glucose uptake rates were decreased. Thus maternal hyperglycemia causes a time-dependent decline in the entire placental glucose transporter pool (GLUT-1 and GLUT-3). In contrast, maternal hypoglycemia decreases GLUT-1 but not GLUT-3, resulting in a relatively increased GLUT-3 contribution to the placental glucose transporter pool, which could maintain glucose delivery to the placenta relative to the fetus when maternal glucose is low.

GLUT8 is a glucose transporter responsible for insulin-stimulated glucose uptake in the blastocyst.

Mammalian preimplantation blastocysts exhibit insulin-stimulated glucose uptake despite the absence of the only known insulin-regulated transporter, GLUT4. We describe a previously unidentified member of the mammalian facilitative GLUT superfamily that exhibits approximately 20-25% identity with other murine facilitative GLUTs. Insulin induces a change in the intracellular localization of this protein, which translates into increased glucose uptake into the blastocyst, a process that is inhibited by antisense oligoprobes. Presence of this transporter may be necessary for successful blastocyst development, fuel metabolism, and subsequent implantation. Moreover, the existence of an alternative transporter may explain examples in other tissues of insulin-regulated glucose transport in the absence of GLUT4.

Distribution of glucose transporter isoform-3 and hexokinase I in the postnatal murine brain.

The facilitative glucose transporter-3 (GLUT 3) and hexokinase I were examined in postnatal mouse brains using immunohistochemical methods. GLUT 3 demonstrated a polarized distribution limited to neuronal processes of most anatomical regions except the suprachiasmatic nucleus and the cerebellum, where GLUT 3 expression was limited to neuronal cell somata. In contrast, hexokinase I was observed in the cytoplasm of neuronal and non-neuronal (subependymal and choroid plexus epithelial) cell bodies in all regions. In general, while the spatial distribution of GLUT 3 and hexokinase I did not change with age, a temporal increase in intensity was noted in all regions except for the decline in suprachiasmatic nuclear GLUT 3 immunoreactivity.

Effect of primary congenital hypothyroidism upon expression of genes mediating murine brain glucose uptake.

Using hyt/hyt mice that exhibit naturally occurring primary hypothyroidism (n = 72) and Balb/c controls (n = 66), we examined the mRNA, protein, and activity of brain glucose transporters (Glut 1 and Glut 3) and hexokinase I enzyme at various postnatal ages (d 1, 7, 14, 21, 35, and 60). The hyt/hyt mice showed an age-dependent decline in body weight (p < 0.04) and an increase in serum TSH levels (p < 0.001) at all ages. An age-dependent translational/posttranslational 40% decline in Glut 1 (p = 0.02) with no change in Glut 3 levels was observed. These changes were predominant during the immediate neonatal period (d 1). A posttranslational 70% increase in hexokinase enzyme activity was noted at d 1 alone (p < 0.05) with no concomitant change in brain 2-deoxy-glucose uptake. This was despite a decline in the hyt/hyt glucose production rate. We conclude that primary hypothyroidism causes a decline in brain Glut 1 associated with no change in Glut 3 levels and a compensatory increase in hexokinase enzyme activity. These changes are pronounced only during the immediate neonatal period and disappear in the postweaned stages of development. These hypothyroid-induced compensatory changes in gene products mediating glucose transport and phosphorylation ensure an adequate supply of glucose to the developing brain during transition from fetal to neonatal life.

Intra-uterine growth restriction differentially regulates perinatal brain and skeletal muscle glucose transporters.

Employing Western blot analysis, we investigated the effect of maternal uterine artery ligation causing uteroplacental insufficiency with asymmetrical intrauterine growth restriction (IUGR) upon fetal (22d) and postnatal (1d, 7d, 14d and 21d) brain (Glut 1 and Glut 3) and skeletal muscle (Glut 1 and Glut 4) glucose transporter protein concentrations. IUGR was associated with a approximately 42% decline in fetal plasma glucose (p<0.05) and a approximately 25% decrease in fetal body weights (p<0.05) with no change in brain weights when compared to the sham operated controls (SHAM). In addition, IUGR caused a approximately 45% increase in fetal brain Glut 1 (55 kDa) with no change in Glut 3 (50 kDa) protein concentrations. This fetal brain Glut 1 change persisted, though marginal, through postnatal suckling stages of development (1d-21d), with no concomitant change in brain Glut 3 levels at day 1. In contrast, in the absence of a change in fetal skeletal muscle Glut 1 levels (48 kDa), a 70% increase was observed in the 1d IUGR with no concomitant change in either fetal or postnatal Glut 4 levels (45 kDa). The change in skeletal muscle Glut 1 levels normalized by d7 of age. We conclude that IUGR with hypoglycemia led to a compensatory increase in brain and skeletal muscle Glut 1 concentrations with a change in the brain preceding that of the skeletal muscle. Since Glut 1 is the isoform of proliferating cells, fetal brain weight changes were not as pronounced as the decline in somatic weight. The increase in Glut 1 may be protective against glucose deprivation in proliferating fetal brain cells and postnatal skeletal myocytes which exhibit 'catch-up growth', thereby preserving the specialized function mediated by Glut 3 and Glut 4 towards maintaining the intracellular glucose milieu.

Effect of development and hypoxic-ischemia upon rabbit brain glucose transporter expression.

We have cloned and sequenced a full length rabbit GLUT 1 and partial rabbit GLUT 3 cDNAs. The derived rabbit GLUT 3 peptide revealed 84% homology to the mouse, 82% to the rat, human, dog, and sheep, and 69% to the chicken GLUT 3 peptides. Using Northern blot analysis, we investigated the tissue and brain cellular distribution of GLUT 1 and GLUT 3 expression. In addition, we examined the effect of development and hypoxic-ischemia upon brain GLUT 1 and GLUT 3 mRNA levels. While GLUT 1 mRNA was observed in most tissues, GLUT 3 was expressed predominantly in the brain, placenta, stomach, and lung with minor amounts in the heart, kidney and skeletal muscle. In the brain, both GLUT 1 and GLUT 3 were noted in neuron- and glial-enriched cultures. Both GLUT 1 and GLUT 3 mRNA levels demonstrated a similar developmental progression (p<0.05) secondary to post-transcriptional mechanisms. Further, while hypoxic-ischemia did not significantly affect brain GLUT 1 mRNA and protein, it altered GLUT 3 mRNA levels in a region-specific manner, with a three-fold increase in the cerebral cortex, a two-fold increase in the hippocampus, and a 50% increase in the caudate nucleus (p<0.05). We conclude, that the rabbit GLUT 3 peptide sequence exhibits 82-84% homology to that of other species in the coding region with a 62-89% sequence identity in the 3'-untranslated region. The tissue-specific expression of rabbit GLUT 3 mimics that of the human closely. Postnatal development and hypoxic-ischemia with reperfusion injury cause an increase in brain GLUT 3 expression, as a response to synaptogenesis and substrate deprivation, respectively.

Time-dependent and tissue-specific effects of circulating glucose on fetal ovine glucose transporters.

To determine the cellular adaptations to fetal hyperglycemia and hypoglycemia, we examined the time-dependent effects on basal (GLUT-1 and GLUT-3) and insulin-responsive (GLUT-4) glucose transporter proteins by quantitative Western blot analysis in fetal ovine insulin-insensitive (brain and liver) and insulin-sensitive (myocardium, skeletal muscle, and adipose) tissues. Maternal glucose infusions causing fetal hyperglycemia resulted in a transient 30% increase in brain GLUT-1 but not GLUT-3 levels and a decline in liver and adipose GLUT-1 and myocardial and skeletal muscle GLUT-1 and GLUT-4 levels compared with gestational age-matched controls. Maternal insulin infusions leading to fetal hypoglycemia caused a decline in brain GLUT-3, an increase in brain GLUT-1, and a subsequent decline in liver GLUT-1, with no significant change in insulin-sensitive myocardium, skeletal muscle, and adipose tissue GLUT-1 or GLUT-4 concentrations, compared with gestational age-matched sham controls. We conclude that fetal glucose transporters are subject to a time-dependent and tissue- and isoform-specific differential regulation in response to altered circulating glucose and/or insulin concentrations. These cellular adaptations in GLUT-1 (and GLUT-3) are geared toward protecting the conceptus from perturbations in substrate availability, and the adaptations in GLUT-4 are geared toward development of fetal insulin resistance.

Developmental regulation of genes mediating murine brain glucose uptake.

We examined the molecular mechanisms that mediate the developmental increase in murine whole brain 2-deoxyglucose uptake. Northern and Western blot analyses revealed an age-dependent increase in brain GLUT-1 (endothelial cell and glial) and GLUT-3 (neuronal) membrane-spanning facilitative glucose transporter mRNA and protein concentrations. Nuclear run-on experiments revealed that these developmental changes in GLUT-1 and -3 were regulated posttranscriptionally. In contrast, the mRNA and protein levels of the mitochondrially bound glucose phosphorylating hexokinase I enzyme were unaltered. However, hexokinase I enzyme activity increased in an age-dependent manner suggestive of a posttranslational modification that is necessary for enzymatic activation. Together, the postnatal increase in GLUT-1 and -3 concentrations and hexokinase I enzymatic activity led to a parallel increase in murine brain 2-deoxyglucose uptake. Whereas the molecular mechanisms regulating the increase in the three different gene products may vary, the age-dependent increase of all three constituents appears essential for meeting the increasing demand of the maturing brain to fuel the processes of cellular growth, differentiation, and neurotransmission.

Sp1 and Sp3 regulate transcriptional activity of the facilitative glucose transporter isoform-3 gene in mammalian neuroblasts and trophoblasts.

The murine facilitative glucose transporter isoform 3 (Glut 3) is developmentally regulated and is predominantly expressed in neurons and trophoblasts. Employing the primer extension and RNase protection assays, the transcription start site (denoted as +1) of the murine Glut 3 gene was localized to 305 base pairs (bp) 5' to the ATG translation start codon. Transient transfection assays in N2A, H19-7 neuroblasts, and HRP.1 trophoblasts using sequential 5'-deletions of the murine Glut 3-luciferase fusion gene indicated that the -203 to +237 bp region with reference to the transcriptional start site contained promoter activity. Repressor function was limited to the -137 to -130 bp region within the transcriptional activation domain. The nuclear factors Sp1 and Sp3 bound this GC-rich region in N2A, H19-7, and HRP.1 cells. Dephosphorylation of Sp1 was essential for Glut 3 DNA binding. The related Sp3 protein also bound this same region of mouse Glut 3 in all three cell lines. Mutations of the Sp1-binding site employed in transient transfection and mobility shift assays confirmed the nature of the DNA-binding proteins, while supershift assays with anti-Sp1 and anti-Sp3 IgGs characterized the differences in the two DNA-binding proteins. Co-transfection of the Glut 3-luciferase fusion gene with or without mutations of the Sp1-binding site along with the Sp1 or Sp3 expression vectors in Drosophila SL2 cells confirmed a reciprocal effect, with Sp1 suppressing and Sp3 activating Glut 3 gene transcription.

Effect of uteroplacental insufficiency upon brain neuropeptide Y and corticotropin-releasing factor gene expression and concentrations.

Various hypothalamic functions such as feeding behavior, energy expenditure, body weight gain, level of anxiety, and sexual maturation are mediated by a balance between the concentrations of neuropeptide Y (NPY) and corticotropin-releasing factor (CRF). To test the hypothesis that maternal uteroplacental insufficiency alters the offspring's brain NPY and/or CRF levels, we examined the effect of maternal uterine artery ligation with intrauterine growth restriction (IUGR) (p < 0.05) upon fetal (20 d) and postnatal (4, 14, and 21 d) brain NPY and CRF synthesis, concentrations, and regional distribution. An age-related increase in NPY (0.8 kb) and CRF (1.4 kb) mRNA levels with peak amounts at the 14-d postnatal age (p < 0.05) was observed. IUGR was associated with a 75% increase in fetal brain NPY mRNA levels (p < 0.05) with no change in NPY peptide, CRF mRNA and peptide amounts. Although the increase in NPY mRNA levels persisted postnatally (p < 0.05) at d 4 and 21, CRF mRNA amounts were 2.5-fold higher only in the 4-d IUGR (p < 0.05). Paralleling the mRNA changes, an age-related increase in RIA of NPY and CRF peptide concentrations was noted (p < 0.05). IUGR caused postnatal brain NPY and CRF peptide changes similar to corresponding mRNA levels (p < 0.05), despite normal postnatal circulating glucose, insulin, corticosterone, and leptin concentrations. The age-specific intergroup differences in the NPY and CRF peptide immunoreactivity appeared predominantly in the hypothalamic region. We conclude that maternal uteroplacental insufficiency causing IUGR leads to a pretranslational imbalance in the immediate (4 d) postnatal brain NPY and CRF peptide concentrations, thereby altering the developmental pattern. This alteration in NPY and CRF peptide concentrations, despite normalization of the metabolic milieu was associated with a persistent diminution in body weight. The IUGR-associated pretranslational increase in NPY and not CRF peptide levels at d 21, may herald changes in feeding behavior during the postsuckling phase.

Neuropeptide Y gene expression in immortalized rat hippocampal and pheochromocytoma-12 cell lines.

Employing clonal cell lines derived from rat embryonic hippocampal cells, we detected neuropeptide Y (NPY) mRNA in three progenitor subcloned cell lines. These cell lines upon differentiation express markers indicative of commitment to either neuronal (H19-7; NF +, GFAP -), glial (H19-5; GFAP +, NF -), or bipotential (H583-5; NF +, GFAP + ) lineages. Induction of differentiation was associated with the persistence of the NPY mRNA, however, in the differentiated H19-7 cells a 20-fold increase in NPY mRNA levels was observed (P<0.05). NPY immunoreactivity was observed only in cells with a differentiated neuronal phenotype. The cellular radioimmunoassayable NPY peptide levels increased twelve-fold without a change in extracellular NPY peptide levels by multi-factorially induced neuronal or glial cell differentiation. The differentiated H19-5 cells expressed lower levels of NPY that could not be immunocytochemically detected. The peripheral sympathetic PC-12 neuronal cells examined in the undifferentiated and nerve growth factor-driven differentiated states expressed NPY only upon differentiation. We conclude that NPY is expressed by the cultured undifferentiated and differentiated rat hippocampal clonal cell lines, while the peripheral sympathetic PC-12 neuronal cell line only expresses the NPY gene upon differentiation. These immortalized embryonic neural cell line(s) will provide a hippocampal cell line(s) to conduct future in-vitro investigations targeted at determining the cellular and molecular mechanisms governing NPY gene expression.