Cellular energy metabolism during fetal development. II. Fatty acid oxidation by the developing heart.

In view of the importance of fatty acids as substrates for the mature heart, fatty acid oxidation by fetal and calf heart mitochondria has been investigated. Free fatty acids of 10 carbon units or less which exhibit carnitine-independent transport into mitochondria were effective substrates for oxidative phosphorylation in both fetal and calf heart mitochondria. Efficient oxidative phosphorylation with these substrates was dependent upon the presence of bovine serum albumin in the assay medium to reverse the uncoupling effects of the fatty acids. In the presence of bovine serum albumin, ADP/0 ratios were in the range of 3 when short-chain fatty acids and carnitine esters of short- and long-chain fatty acids were substrates. Compared with calf heart mitochondria, fetal heart mitochondria showed decreased carnitine-dependent oxidation of palmityl-CoA. However, the oxidation of palmitylcarnitine was identical in both. These data suggest that the formation of palmitylcarnitine is rate limiting for palmityl-CoA oxidation by the fetal heart mitochondria and that long-chain fatty acids are not readily oxidized by the fetal heart.

Interaction of fatty acid and glucose oxidation by cultured heart cells.

Chick embryo heart cells in tissue culture actively oxidize [1-(14)C]palmitate to (14)CO(2). Fatty acid oxidation by cell monolayers was linear with time and increasing protein concentration. The addition of carnitine to the assay medium resulted in a 30-70% increase in the rate of fatty acid oxidation. The specific activity of palmitic acid oxidation did not change significantly with time in culture and was also the same in rapidly proliferating and density-inhibited cell cultures. Addition of unlabeled glucose to the assay medium resulted in a 50% decrease in (14)CO(2) production from [1-(14)C]palmitate. Conversely, palmitate had a similar sparing effect on [(14)C]glucose oxidation to (14)CO(2). Lactate production accounted for most of the glucose depleted from the medium and was not inhibited by the presence of palmitate in the assay. Thus, the sparing action of the fatty acids on glucose oxidation appears to be at the mitochondrial level. The results indicate that although chick heart cells in culture are primarily anaerobic, they can oxidize fatty acid actively.

Changes in glucose oxidation during growth of embryonic heart cells in culture.

A rapid and convenient method has been utilized to investigate glucose oxidation during growth of chick embryo heart cells in tissue culture. Primary isolates of chick embryo heart cells showed exponential growth when plated at low densities and exhibited density-inhibited growth as cultures became confluent. The density-dependent growth inhibition of chick embryo heart cells is associated with a marked decrease in the specific activity of glucose oxidation to CO(2). This decrease in glucose oxidation was observed as density increased as either a function of time in culture or as related to initial plating density. The decrease in (14)CO(2) production associated with density-dependent inhibition of growth is due to a marked decrease in activity of the pentose phosphate pathway.

Endocrine studies in anencephaly.

Endocrine function has been investigated in four anencephalic neonates to determine the influence of absence of cortical and hypothalamic tissue and of hypoplasia of the pituitary. Intravenous glucose administration resulted in higher peak values for blood sugar and more rapid glucose disposal rates than reported in normals. Intravenous insulin tolerance tests on two of the infants failed to evoke elevations in plasma growth hormone, and the infants showed a remarkable resistance to the hypoglycemic effect of insulin. Administration of lysine-vasopressin caused an active growth hormone release. Similarly, there was a large increase in serum thyrotropin after administration of synthetic thyrotropin-releasing hormone. Basal levels of both thyrotropin and growth hormone were low as compared with values reported for normal newborns. Prolactin values obtained on three of the infants were in the normal range. The results strongly suggest that anterior pituitary function mediated by the hypothalamus and its releasing factors is deficient in anencephaly. However, the anterior pituitary can release growth hormone and thyrotropin when stimulated directly and, in the case of thyrotropin release, may function autonomously. The normal prolactin values presumably reflect the absence of the hypothalamic prolactin inhibitory factor.

Decreased binding of epidermal growth factor in placentas from streptozotocin-diabetic rats.

Placentas from streptozotocin-diabetic rats have previously been shown to be morphologically and biochemically immature when compared with those of control rats. The binding of epidermal growth factor (EGF) to plasma membranes prepared from placentas of control and streptozotocin-diabetic fetuses has been characterized on days 17 and 21 of gestation. Results from competitive binding data analyzed by Scatchard analysis indicate the presence of a single class of receptors on day 17 (KD = 5.4 X 10(-10)) and the appearance of a second class of binding sites for 125I-EGF by day 21 (Kd = 3.5 X 10(-9)) in membranes from control fetuses. Placental membranes from diabetic fetuses show decreased specific binding (approximately 30%) on both days and the absence of a second class of binding sites on day 21 of gestation. Results from a radioreceptor assay indicate that the quantity of EGF in the serum of fetuses removed from control rats on day 21 is twofold greater than the quantity in serum of fetuses from diabetic rats. These data reveal a developmental increase in EGF-binding sites in the placenta of normal, near-term fetal rats, largely because of the appearance of a second class of binding sites with a lower affinity for EGF. The failure (or delay) of this second class to develop in the diabetic may be important for the control of maturation and growth of this tissue.

Effects of maternal diabetes on fetal rat lung ion transport. Contribution of alveolar and bronchiolar epithelial cells to Na+,K(+)-ATPase expression.

Fetuses of streptozotocin-induced diabetic rats exhibited delayed lung maturation and a 40% reduction in the steady-state level of lung Na+,K(+)-ATPase alpha 1 subunit mRNA and Na+,K(+)-ATPase activity at 21 d of gestation. In in situ hybridization experiments the signal specific for Na(+)-pump alpha 1 subunit message was strongest above columnar epithelial cells of air-conducting structures. Strong labeling was also present above cuboidal cells lining the forming alveoli, but not above mesenchymal cells. Immunocytochemical localization of the protein paralleled the distribution of the mRNA. Mesenchymal cells were more abundant in fetal lungs of diabetic mothers, and thus the decreased overall levels of Na+,K(+)-ATPase may result from the observed morphological pulmonary immaturity. One day after birth there was no apparent difference in lung morphology at the light microscopic level, in the localization or the steady-state level of Na+,K(+)-ATPase alpha 1 isoform mRNA, or in enzyme activity. Na+,K(+)-ATPase has a likely role in the active phase of fluid absorption in the airways of newborns before the onset of breathing. Decreased fluid clearance and lack of thinning of the lung's connective tissue may contribute to the increased risk for respiratory distress in infants of diabetic mothers.

Serum alkaline phosphatase in hypophosphatasia.

It is recognized that serum alkaline phosphatase may reflect enzyme contributions from bone, liver, and intestine. We have investigated serum alkaline phosphatases in two siblings with hypophosphatasia. After administration of long-chain triglycerides, the major alkaline phosphatase component of their sera was shown to be of intestinal origin on the basis of inhibition by l-phenylalanine. Starch block electrophoresis suggested that there were other regions of l-phenylalanine-sensitive alkaline phosphatase in addition to the major slow-moving intestinal band. Medium-chain triglycerides which are absorbed by the portal route did not cause a similar augmentation of intestinal alkaline phosphatase activity. These studies indicate that serum levels of intestinal alkaline phosphatase are increased normally after long-chain fat feeding in hypophosphatasia and may be the major component of total serum alkaline phosphatase activity.

Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation.

Acute lung injury is a frequent and treatment-limiting consequence of therapy with hyperoxic gas mixtures. To determine if IL-11 is protective in oxygen toxicity, we compared the effects of 100% O2 on transgenic mice that overexpress IL-11 in the lung and transgene (-) controls. IL-11 markedly enhanced survival in 100% O2 with 100% of transgene (-) animals dying within 72-96 h and > 90% of transgene (+) animals surviving for more than 10 d. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Significant differences in copper zinc superoxide dismutase and catalase activities were not noted and the levels of total, reduced and oxidized glutathione were similar in transgene (+) and (-) animals. Glutathione reductase, glutathione peroxidase, and manganese superoxide dismutase activities were slightly higher in transgene (+) as versus (-) mice after 100% O2 exposure, and IL-11 diminished hyperoxia-induced expression of IL-1 and TNF. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals and IL-11 markedly diminished this cell death response. These studies demonstrate that IL-11 markedly diminishes hyperoxic lung injury. They also demonstrate this protection is associated with small changes in lung antioxidants, diminished hyperoxia-induced IL-1 and TNF production, and markedly suppressed hyperoxia-induced DNA fragmentation.

Development of glycogen and phospholipid metabolism in fetal and newborn rat lung.

Glucose, a major metabolic substrate for the mammalian fetus, probably makes significant contributions to surface active phospholipid synthesis in adult lung. We examined the developmental patterns of glycogen content, glycogen synthase activity, glycogen phosphorylase activity and glucose oxidation in fetal and newborn rat lung. These patterns were correlated with the development of phosphatidylcholine synthesis, content and the activities of enzymes involved in phosphatidylcholine synthesis. Fetal lung glycogen concentration increased until day 20 of gestation (term is 22 days) after which it declined to low levels. Activity of both glycogen synthase I and total glycogen synthase (I + D) in fetal lung increased late in gestation. Increased lung glycogen concentration preceded changes in enzyme activity. Glycogen phosphorylase a and total glycogen phosphorylase (a + b) activity in fetal lung increased during the period of prenatal glycogen depletion. The activity of the pentose phosphate pathway, as measured by the ratio of CO2 derived from oxidation of C1 and C6 of glucose, declined after birth. Fetal lung total phospholipid, phosphatidycholine and disaturated phosphatidylcholine content increased by 60, 90 and 180%, respectively, between day 19 of gestation and the first postnatal day. Incorporation of choline into phosphatidylcholine and disaturated phosphatidylcholine increased 10-fold during this time. No changes in phosphatidylcholine enzyme activities were noted during gestation, but both choline phosphate cytidylyltransferase and phosphatidate phosphatase activity increased after birth. The possible contributions of carbohydrate derived from fetal lung glycogen to phospholipid synthesis are discussed.

Fine structural abnormalities of the placenta in diabetic rats.

In the streptozocin-induced diabetic rat, the placenta is larger and the fetus is smaller than normal. To study cellular differences that might contribute to the size and functional disparity between diabetic and control placentas, a light- and electron-microscopic analysis was performed on 14-, 18-, and 22-day (term) control and diabetic placentas. Diabetic placentas, especially later in gestation, were marked by the presence of large numbers of glycogen-distended cells in the basal zone. Within the placental labyrinth, the trophoblastic layers of the interhemal membrane were significantly thicker in the diabetic placentas on days 18 and 22, and large accumulations of liid droplets were present, especially in the inner two trophoblastic layers. In normal placentas there is a marked thinning of the placental barrier in the labyrinth by day 22 of gestation, making the thickness of the labyrinthine layers in age-matched diabetic placentas even more impressive. Finally, the labyrinth of 22-day diabetic placentas contained more glycogen and rough endoplasmic reticulum in the inner trophoblastic layer, a feature that is found in less-mature (18-day) control placentas. Thus, the diabetic placentas have a number of features that are consistent with functional immaturity/dysmaturity. Cytologic evidence confirms the presence of increased glycogen and lipid reserves in both the junctional zone and the cellular area between maternal and fetal blood.(ABSTRACT TRUNCATED AT 250 WORDS)

Relevance of the gestational age distribution of meconium passage in utero.

The incidence of meconium staining of the amniotic fluid among the admissions to a newborn special care unit was determined. Meconium staining of the amniotic fluid was present in 251 (7.5%) of 3,374 admissions. The majority of those with meconium staining (98.4%) was of 37 weeks gestation or greater and none was less than 34 weeks gestation. The possible relevance of this gestational age distribution is discussed.

Importance of radiant flux in the treatment of hyperbilirubinemia: failure of overhead phototherapy units in intensive care units.

Prior to 1972, radiation used to treat neonatal hyperbilirubinemia was based upon the photometric unit, the foot-candle, a measure of light illumination. Measurements in terms of microwatts per square centimeter for selective wavelengths is more precise. We compared the effectiveness of phototherapy provided by overhead phototherapy units in intensive care modules vs. conventional phototherapy units. Forty-two infants were studied over a six-month period and divided into three groups based upon radiant flux measurements as follows: Group 1 (No. = 6), 1.0 muw to 1.9 muw/sq cm/nm; group 2 (No. = 15), 2.0 muw to 3.9 muw/sq cm/nm; group 3 (No. = 21), 4.0 muw to 6.0 muw/sq cm/nm. All flux determinations were made within the 400- to 500-nm range. All infants in group 1 were treated with overhead phototherapy units in the intensive care modules. Because of multiple factors known to increase the risk of kernicterus, evaluation of effectiveness of phototherapy at low radiant flux was limited in group 1. Significant changes in bilirubin were noted by 48 hours when comparing group 3 with groups 1 and 2. A minimum of 4.0 muw/sq cm/nm appears necessary for effective phototherapy. As designed, phototherapy units in intensive care modules are ineffective in delivering this therapeutic level of radiant flux.

A comparison of fluorescent and nonfluorescent light sources for phototherapy.

We have compared fluorescent and nonfluorescent light sources for phototherapy for newborn infants with hyperbilirubinemia. Phototherapy was provided by a tungsten halogen lamp and conventional fluorescent light with identical radiant flux of 6 microW/sq cm. For 22 infants treated with the nonfluorescent lamp the mean duration of phototherapy was 33.77 hours and the mean reduction of bilirubin was 3.84 mg/100 ml/day. This did not differ significantly from infants treated with conventional fluorescent lights. The nonfluorescent light can be utilized for infants in incubators or on radiant warmers. These results provide additional support for the relationship between radiant flux as a practical measure of phototherapy dose and the clinical response of a reduction in serum bilirubin.

Treatment of severe apnea in prematures with orally administered theophylline.

Twelve premature infants with primary apnea were treated with theophylline as an alternative to mechanical ventilation. There was a significant (P smaller than .005) reduction in the mean daily number and the severity of apneic episodes after treatment. The only significant side effect noted was a rise in heart rate.

Rat lung antioxidant enzyme activities and their specific proteins during hyperoxia.

The hyperoxia-induced increases in the activity of lung glucose-6-phosphate dehydrogenase (G-6-P) and glutathione reductase (GR) after exposure of rats to greater than 97% O2 for 6 days were accompanied by equivalent increases in the amount of the respective immunoreactive proteins. Hyperoxia also increased lung glutathione (GSH) + oxidized glutathione (GSSG) content and the magnitude of this hyperoxic response of increased GSH + GSSG, G-6-P, and GR (maximal 1.3- to 1.8-fold) declined as a function of age during the first 3 wk of life. Fetal rat lung explants cultured 4 days in 95% O2 showed increased G-6-P and GR activity and increased levels of the specific proteins 1.5-fold those of explants at 2 days of culture. We conclude that the hyperoxic response of increased rat lung G-6-P and GR activity in vivo and in vitro involves not just alteration of enzyme activity but also specific increases in the proteins catalyzing the reactions.

Chronic hypoxia causes opposite effects on glucose transporter 1 mRNA in mature versus immature rat brain.

We have shown previously that chronic hypoxia can regulate the expression of membrane proteins. Since there are virtually no glucose stores in the brain and glucose transport can be rate-limiting during stress, the role of glucose transporters becomes crucial for cell survival under stress. In the present study, we asked whether mRNA levels for glucose transporter 1 (GT1), which is expressed in a variety of cells in the brain, especially in the microvessels for glucose transport from blood vessels to brain, change in response to chronic hypoxia. Because major developmental changes occur in the rat CNS in-utero and in the first few weeks postnatally, we studied brain GT1 mRNA using Northern blot analysis at different ages after exposure of fetuses (from embryonic day 10 to birth), developing rats (from birth to 30 day old) or adult rats (from 90 to 120 day old) to hypoxia (Fractional inspired O2 9%). Our data show that (i) GT1 mRNA level was much lower in the newborn than in the adult and increased with age; (ii) chronic hypoxia caused a decrease of approximately 65% in GT1 mRNA in adult brain but induced an increase in fetal (more than 50%) and developing (approximately 80%) rats and (iii) the response of housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase) was not similar to that of GT1, suggesting that the changes of GT1 mRNA are specific to glucose transporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Nutritional perturbations in infants of diabetic mothers and intrauterine growth retardation.

The fetal nutritional milieu may have important regulatory influences on fetal growth and maturation. Fetuses of diabetics exposed to excessive glucose in late gestation show delayed maturation, whereas, fetuses subjected to nutrient deprivation resulting from decreased uterine blood flow exhibit restricted growth and accelerated maturation. Under conditions of nutrient deficiency, restricted growth and accelerated maturation may be important adaptations mediated through hormonal and growth factor signalling.