Uday P. Devaskar

Expression of genes involved in placental glucose uptake and transport in the nonobese diabetic mouse pregnancy

OBJECTIVE Maternal diabetes alters placental glucose metabolism and maternofetal glucose transport. The purpose of this study was to determine whether genes involved in placental glucose uptake and transport were concomitantly altered, resulting in the observed changes in the state of maternal diabetes. STUDY DESIGN By means of the nonobese diabetic pregnant mouse we examined the expression of placental glucose transporters, hexokinase I, glycogen content, glycogen-regulating enzyme activities in control animals (blood glucose 8.5 +/- 0.2 mmol/L, n = 25), moderate maternal diabetes (blood glucose 10 to 13.9 mmol/L, n = 16), and severe maternal diabetes (blood glucose > 16.7 mmol/L, n = 12). Comparisons by the analysis of variance and the Newman-Keuls test were performed. RESULTS Although changes in placental glucose transporters and hexokinase I messenger ribonucleic acid levels occurred, neither state of diabetes altered the corresponding protein levels. Changes in placental deoxyribonucleic acid (p < 0.05) and glycogen content (p < 0.01), fetal insulin levels (p < 0.02), and fetal size (p < 0.05) occurred in the moderately diabetic group, and changes in placental weight (p < 0.05) and fetal glucose levels (p < 0.02) were observed in the severely diabetic group. CONCLUSIONS Placental glucose transporting and phosphorylating protein levels by themselves do not regulate diabetes-induced fetoplacental alterations. The lack of a protective decline in these proteins may account for the observed fetoplacental adaptations to excess glucose.

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.

Hypothyroidism Delays Fetal Stratum Corneum Development in Mice

The epidermal permeability barrier, required for terrestrial life, is localized to lipid-enriched lamellar membranes in the extracellular spaces of the stratum corneum (SC). Immaturity of the SC is a significant contributor to morbidity and mortality in premature infants. Previous studies have shown that supraphysiologic concentrations of thyroid hormone accelerate epidermis/SC ontogenesis. Here we studied SC development in Hyt/Hyt mice who are genetically hypothyroid due to a mutation in the TSH receptor. In control mice on d 18 of gestation (term 19.5 d), only focal areas displayed a mature SC membrane pattern. By 19 d of gestation there was a mature multilayered SC with lamellar unit structures filling the extracellular spaces similar to that seen in mature mice. In Hyt/Hyt mice SC development was delayed at both 18 and 19 d of gestation. In both strains of mice, within the first day after birth there were no differences in epidermal or SC appearance, and the SC was fully mature. These findings indicate that thyroid hormone plays a physiologic role during normal intrauterine development of the SC. However, normal SC maturation ultimately occurs, indicating that thyroid hormone is not absolutely essential. Previous studies have shown that glucocorticoids accelerate SC development in euthyroid rats, and in the present study we demonstrate that glucocorticoids also accelerate SC ontogenesis in euthyroid mice. In contrast, in Hyt/Hyt mice glucocorticoids did not accelerate or normalize SC development, indicating that the glucocorticoid effect on SC maturation requires a euthyroid state or that glucocorticoids act via thyroid hormone. These studies demonstrate that thyroid hormone status is an important regulator of fetal SC development.

Hyperoxia induces interstitial (type I) and increases type IV collagenase mRNA expression and increases type I and IV collagenolytic activity in newborn rat lung

Oxygen toxicity is attributed to the reaction of oxygen metabolites with cellular components leading to cell destruction. Activation of latent human neutrophil interstitial collagenase by reactive oxygen species has been demonstrated. The potential role of collagenases in hyperoxic lung injury has not been investigated. We studied the effect of hyperoxia on newborn rat lung water content, morphology and ultrastructure, interstitial (type I) and type IV collagenase gene expression and type I and IV collagenolytic activity. We observed that hyperoxia causes pulmonary edema, alters newborn rat lung morphology in a sequential manner and produces ultrastructural alterations, induces type I and increases type IV collagenase mRNA expression, and increases type I and IV collagenolytic activity. A role for type I and IV collagenase in hyperoxic newborn lung injury or in the recovery following the injury is proposed.

Transplacental stimulation of functional and morphologic fetal rabbit lung maturation: Effect of thyrotropin-releasing hormone

We investigated the effect of maternally administered thyrotropin-releasing hormone on functional and morphologic fetal lung maturation. Thyrotropin-releasing hormone (40 micrograms/kg/day) or the vehicle was injected intravenously into the New Zealand White rabbit does on days 25, 26, and 27 of gestation. On day 27 of pregnancy, the does were killed and the fetuses were delivered. The functional pulmonary maturity was assessed by pressure-volume hysteresis while morphologic maturity was assessed by histologic techniques. Enhanced functional and morphologic fetal lung maturation was noted in animals treated with thyrotropin-releasing hormone when compared with controls. An important role of thyrotropin-releasing hormone in fetal lung maturation is proposed.

Glucocorticoids increase pulmonary Epidermal Growth Factor receptors in female and male fetal rabbit

Experimental evidence in animals and humans suggest that glucocorticoids enhance fetal pulmonary maturation. Mechanisms of glucocorticoid effects remain unclear; but apparently include up regulation of fetal pulmonary insulin and beta-adrenergic receptors. A role of Epidermal Growth Factor (EGF) in fetal lung maturation through plasma membrane bound receptors has been recently proposed. Betamethasone, 0.085 mg/kg, was administered on 25th and 26th day of gestation to the rabbit doe. Fetal pulmonary EGF receptor characteristics in male or female fetuses were studied on the 27th day of pregnancy. The percent specific binding of 125-I-EGF to lung plasma membranes (LPM) and the number of receptor sites per mg of LPM protein or DNA content were significantly higher in the glucocorticoid treated female as well as male fetuses when compared to the control pups, with no difference in the Kd. Presence of high affinity receptors for EGF and their up regulation by glucocorticoids support the hypothesis that EGF plays an important role in fetal lung maturation and that some of the beneficial effects of glucocorticoids in decreasing the incidence of HMD may be mediated through its interaction with EGF.

Epidermal growth factor receptors in fetal and maternal rabbit lung.

The pattern of morphologic and functional development of lung during intrauterine period is influenced by several endogenous compounds. Recently Epidermal Growth Factor (EGF), when administered in vivo, has been shown to accelerate pulmonary maturation in fetal rabbit and sheep. We sought evidence for EGF receptor occurrence in fetal and maternal rabbit lung plasma membranes. The percent specific binding (mean ± S.E.M.) (125-I) EGF to LPM in the mother (n=5) and the fetus at term (n=7) was 1.08 ± 0.08 and 2.25 ± 0.12 per 175 μg of LPM protein respectively. The number of receptor sites per mg of LPM protein in the mother were significantly less than that in the fetus (44 ± 11 and 250 ± 24 × 10−10, p < 0.001) with no apparent differences in Kd (2.10 ± 0.39 and 2.47 ± 0.24 × 109). Presence of high affinity receptors for EGF in fetal and maternal lung plasma membranes suggests a direct role of EGF in fetal lung maturation.

Effect of Maternal Diabetes on the Expression of Genes Regulating Fetal Brain Glucose Uptake

Diabetes alters adult brain glucose uptake and glucose transporter 1 gene expression. To investigate the effect of diabetes on genes regulating fetal brain glucose uptake, we examined the effect of moderate (blood glucose 10–16.7 mM, normoinsulinemia) and severe (blood glucose >16.8 mM, hypoinsulinemia) maternal diabetes on the expression of genes regulating fetal brain glucose uptake in the genetically nonobese diabetic mouse. In the moderately diabetic state, a 50% decline in fetal brain GLUT1 mRNA levels was associated with a 20% increase in the corresponding GLUT1 protein levels. Simultaneously, although fetal brain GLUT3 mRNA and protein levels were barely detectable, no change in hexokinase I enzyme mRNA, protein (115,000 and 100,000 Mr) or activity, was noted. In the severe form of maternal diabetes GLUT1 protein was unchanged, GLUT3 protein levels remained low, and a 2- to 3-fold increase in the lower molecular form of the hexokinase I protein (100,000 Mr) and enzyme activity occurred. These observations suggest that moderate and severe forms of maternal diabetes do not affect the fetal brain glucose transporter levels to a physiologically significant extent. The severe form of maternal diabetes, however, enhances 1.5- to 3-fold the expression and activity of hexokinase I. This enzyme mediates the rate-limiting step in brain glucose metabolism, namely the intracellular conversion of glucose to glucose–6-phosphate.

Varying brain insulin concentrations differentially regulate the fetal brain insulin receptor

We investigated the downregulating effect of varying states (physiologic and pharmacologic) of systemic and intracranial hyperinsulinism on the 28 to 30 day fetal rabbit brain insulin receptor. Alloxan-induced maternal diabetes (n = 5) produced mild fetal hyperinsulinemia (D) (plasma insulin concentrations = 59.80 +/- 8.10 microU/ml, control = 26.25 +/- 3.70; p less than 0.01), whereas systemic administration (IMI) of 1.0 U (n = 4) and 2.0 U (n = 4) of insulin to the fetus resulted in moderate (103.13 +/- 34.63 microU/ml) and severe (288.3 +/- 51 microU/ml) fetal hyperinsulinemia respectively. All three states of systemic hyperinsulinemia neither altered the fetal brain insulin content nor the brain insulin receptor number and affinity. 0.01 U (n = 4) of intracranial insulin administration (ICI) increased the brain insulin content four-fold (p less than 0.01) but did not alter the brain insulin receptor number or affinity. 0.1 (n = 5) and 2.0 U (n = 7) of intracranial insulin increased the brain insulin content to supraphysiologic concentrations (p less than 0.01) and decreased the fetal brain insulin receptor number (p less than 0.01), the affinity remaining constant. We conclude that 1) regardless of the ability of insulin to cross the blood brain barrier, the downregulation of the brain insulin receptor is insulin dose-dependent and 2) the downregulation of the fetal brain insulin receptor is not a physiologic but a pharmacologic effect of insulin.

Glut 1-glucose transporter protein in adult and fetal mouse lung

We observed approximately 45-50 kD GLUT 1 protein in mouse lung homogenates and demonstrated a greater abundance in fetus compared to adult. In situ immunohistochemical analysis demonstrated GLUT 1 expression only in the perineural sheath of nerves. While the trapped fetal red blood cells expressed GLUT 1 abundantly, adult red blood cells were devoid of GLUT 1. No GLUT 1 was evident in fetal and adult lung alveolar and bronchiolar epithelial cells, vascular endothelial cells and the lung mesenchymal elements. Thus, GLUT 1 is not the major lung glucose transporter.