Rosario A. Rajakumar

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.

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.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.

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.

Prenatal Glucocorticoid Induced Fetal and Postnatal Growth Restriction is Mediated by Obese Gene (OB) or Leptin Receptors † 1559

Prenatal glucocorticoid (GC) therapy is an accepted practice in the prevention of respiratory distress syndrome in the neonate. Concerns regarding its use in early gestation and its side effects upon fetal and postnatal growth and metabolism persist. The recent cloning of the obese gene and obese gene receptors (OB-R) has shed some light upon mechanisms by which altered metabolism (anorexic/catabolic) and diminution in growth occurs. To determine if the ob gene (leptin) receptors mediate the growth reducing effects of GC, we intraperitoneally administered 2 doses of 0.3 mg/kg/day dexamethasone (GC) or vehicle (V) beginning d7, d9, d11, d13, d15, d17, and d19 (term ≈21d; n=7/age/group), and examined the binding of125 I-leptin (10-9M), in the presence and absence of 0.6 X 10-6M unlabeled leptin, to the d9, d11, d13, d15, d17, d19, and d21 frozen saggital embryonic liver sections. In addition, postnatal livers were studied on d15 and d30 (n=7/age/group) subsequent to GC or V administration on d19 gestation. Specific 125I-leptin binding was assessed by quantitation of the autoradiographs and phoshoimages by microdensitometry(Molecular Analyst Microdensitometry Program-1.4.1, NIH). GC reduced body weight at all ages (p < 0.001), and increased hepatic 125I-leptin binding at all ages with a maximal 3-fold increase noted at postnatal d15 (p< 0.005). This is in contrast to a 2-fold decline in hepatic125 I-insulin binding (p < 0.01) at postnatal d15, which served as a known control. We conclude that prenatal GC 1] increases hepatic leptin receptors from mid- to late gestation, 2] has a persistent enhancing effect upon postnatal hepatic leptin receptors. We speculate that prenatal GC increases the biological action of leptin peripherally, thereby affecting the metabolic mileu and the growth potential of the fetus and the newborn.

Nuclear Factor(s) Regulating the Developmental Changes in Mouse Brain Glucose Transporter (Glut 3) Expression † 472

Nuclear Factor(s) Regulating the Developmental Changes in Mouse Brain Glucose Transporter (Glut 3) Expression † 472

Obese Gene (Leptin) Receptors are Widely Distributed in Embryonic Tissues • 293

Leptin receptors (LR) mediate central (satiety) and peripheral metabolic actions of the translated and secreted product of the obese (ob) gene. While two isoforms as products of RNA splicing have been characterized, both of them bind leptin on the same epitope. Even though LR has been reported in adult tissues, limited information exists with respect to its presence and tissue-distribution in the embryo. We examined the spatial and temporal distribution of LR in the rat conceptus and placenta from d9 to d21 at 2 day intervals (term≈d21), and postnatal d15, and d30 (n=7/age/group). Employing frozen saggital embryonic and postnatal rat sections, 125I-leptin binding in the presence and absence of 0.6 × 10-6M unlabeled leptin was assessed by autoradiography and microdensitometry (Molecular Analyst Microdensitometry Program - 1.4.1, NIH). On d9, the placenta revealed LR while the known control (125I-insulin binding [IR]) was noted in amniotic membranes. At d11, LR was noted in the liver, while liver IR was present at d13. LR was present in the brain [d13], spine and long bones [d13], heart [d15], and kidney [d17]. The LR increased 2.5 fold in density from d9 to d21 (p < 0.01), with a subsequent 3-fold rise postnatally reaching a peak at d15 (p < 0.05). In contrast, IR demonstrated a 1.5-fold increase prenatally followed by a peak at postnatal d15. We conclude that 1] LR are present in embryonic tissues although the ontogenic peak is observed postnatally at d15, 2] LR are distributed in various tissues, with the earliest appearance being limited to the placenta and liver even before its appearance in the brain. We speculate that LR mediate the biological effects of leptin both centrally and peripherally in the embryo thereby regulating metabolism and growth.

Primary hypothyroidism alters expression and function of the hexokinase I gene which mediates brain glucose phosphorylation. † 1390

Primary hypothyroidism alters expression and function of the hexokinase I gene which mediates brain glucose phosphorylation. † 1390

Sp1 nuclear factor represses glucose transporter (Glut 3) gene transcription in mammalian neurons and placental trophoblasts. |[bull]| 412

Glut 3, the most efficient facilitative glucose transporter, is solely expressed in murine brain neurons (N) and placental trophoblasts (T), and ensures glucose supply to fuel neurotransmission and fetal growth. To determine the molecular mechanisms which control Glut 3 expression in N and T, we undertook primer extension assays and established the transcription start site (TSS) of the mouse Glut 3 gene upstream from the ATG translation start codon at -305 bp in the N2A murine neuroblasts, and at -125 bp in the HRP.1 rat placental T. We then cloned the -203 to +243 bp (numbered per the TSS in N) mouse Glut 3 DNA which contains putative binding sites and successive 5′-deletions, upstream to a promoterless and enhancerless luciferase reporter gene. These deletional constructs were transfected into the N2A, and HRP.1 cells and the reporter gene activity assessed. The -203 to +243 bp DNA exhibited an increase (promoter), where as the -177 to +243 bp DNA showed an inhibition of the reporter gene activity (repression) which disappeared in the-104 to +243 bp DNA fragment. Mobility shift (EMSA) and DNAse 1 footprinting assays established that the -143 to -123 bp mouse Glut 3 DNA duplex specifically bound a nuclear protein from N2A and HRP.1 cells. Computer analysis for potential DNA binding motifs and competition EMSA using oligonucleotides with a consensus sequence for canonical transcription factors suggested, while supershift assays established the nuclear factor Sp1 to bind this DNA element. We conclude that 1] alternate TSS situated ≈180 bp apart initiate Glut 3 transcription in N vs. T, and 2] Sp1 represses the promoter-driven Glut 3 transcriptional activity in N and T. We speculate that changes in Sp1 DNA-binding activity modulate Glut 3 gene expression and function in N and T.

Brain Hexokinase I Expression and Function during Development. † 1391

Glucose, an essential substrate for brain growth, cellular maturation, and oxidative metabolism is transported across the blood-brain barrier, into neurons and glial cells. Intracellularly glucose is phosphorylated into glucose-6-phosphate by the hexokinase I (hxl) enzyme. In the adult brain, glucose phosphorylation comprises the rate-limiting step in the process of glucose uptake. To determine the ontogeny of this critical rate limiting step, we examined the brain hxl expression (Northern blots), concentration (Western blots), enzymatic activity (NADPNADPH conversion by spectrophotometric assay) and function (3H-2-deoxy-glucose uptake) in Balb-C mice at 1d(n=6), 14d (n=6), and 35d (n=6) postnatal ages. Hxl mRNA and protein concentrations declined 20-50% (p < 0.05) while activity increased 2-fold between the 1d and 14d or 35d brains. In contrast, a six-fold increase in3 H-2-deoxy-glucose uptake (p < 0.05) which quantitates glucose transport and phosphorylation was noted between the 1d and 14d old mice. Our previous investigation demonstrated a 3-fold increase in brain glucose transporter expression and levels (particularly neuronal Glut 3) between the 1d and 14d mouse brains (Ped Res 39:91A, 1996). We conclude that 1] brain Hxl enzyme activity and function peak by a post-translational mechanism at the 14d postnatal age, 2] the age-related increase in brain Hxl enzyme activity along with the previous observation of a parallel increase in brain glucose transporter concentrations substantiate the age-related increase in brain 2-deoxyglucose uptake with a peak at 14d postnatal age. We speculate that this age-dependent increase in the mechanisms mediating brain glucose uptake 1] may be initiated by the physiological surge in thyroid hormone levels and activity that occurs at 14d of age, and 2] is critical for fueling the process of rapid brain growth and cellular development that occurs at 14d postnatal age in preparation for acquiring the specialized function of neurotransmission.

PRIMARY HYPOTHYROIDISM TRANSLATIONALLY REGULATES THE DEVELOPMENT OF BRAIN GLUCOSE TRANSPORTER EXPRESSION. |[dagger]| 532

Glucose, an essential substrate for brain oxidative metabolism, is transported by facilitative glucose transporter proteins (Glut) across the blood brain barrier into glia (Glut 1) and neurons (Glut 3). Since primary hypothyroidism (Hypo-T) adversely affects brain metabolism (myelination) and neurological function, and thyroid hormones (TH-T4/T3) regulate Glut levels in other tissues, we studied the effect of Hypo-T on brain Glut 1 and Glut 3 mRNA and protein levels in Balb-hyt/hyt mice. The Balb-C (+/+) mouse served as the euthyroid control. Hypo-T characterized by a high serum TSH and a low TH concentration occurs due to a point mutation (Pro556-Leu) in theβ-subunit of the TSH receptor and is transmitted as an autosomal recessive trait. Brain homogenates, obtained from 18d fetal (n=6), 1d (n=6), 7d (n=6), 14d (n=6), 21d (n=6), 35d (n=6) and 60d (n=6) postnatal mice were subjected to mRNA extraction, quantitative Northern blot analysis and protein estimation with quantitative Western blot analysis. In Balb-C mice a gradual increase in both Glut 1 and Glut 3 proteins was noted with advancing age, the peak being at 21d (p <0.02) with the value remaining unchanged through 60d. This developmental alteration was paralleled by corresponding mRNA changes (p< 0.01). Comparison of hyt/hyt versus Balb-C mice at 7d (n=6 vs.6), 14d(n=6 vs. 6) and 21d (n=6 vs. 6) postnatal ages revealed changes in brain Glut 1 (decreased to 50%, p < 0.05) and Glut 3 (increased to 60%, p < 0.05) proteins. These developmental changes were not reflected in the corresponding mRNA levels. We conclude that 1] developmental changes in brain Glut 1 and Glut 3 proteins are regulated at a pretranslational level, and 2] Hypo-T alters brain Glut 1 and Glut 3 protein concentrations at a translational or post-translational level. While brain glucose uptake studies are ongoing, we speculate that these reciprocal changes in brain Glut 1 and Glut 3 proteins may help preserve neuronal glucose metabolism and function, while adversely affecting glial metabolism and myelination.