Christine M. Wilson

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.

Glucocorticoid-Thyroid Hormone Interactions in Fetal Rat Lung

Summary: Previous studies have shown that triiodothyronine (T3) enhances the effect of dexamethasone on phosphatidylcholine (PC) synthesis in organ cultures of fetal rat lung. The aim of this study was to investigate whether similar interactions occurred in vivo and to explore possible mechanisms for this phenomenon.Injection of 7.0 mg/kg T3 into pregnant rats on d 18 and 19 of gestation resulted in a mean fetal serum T3 level of 2380 ng/dl on d 20 (control, 84 ng/dl) and in maximal (34%) stimulation of choline incorporation into PC. Injection of 1.0 mg/kg betamethasone using the same protocol as for T3 resulted in maximal stimulation of 33% and administration of both hormones together produced a 69% increase, an additive affect. The percentage of PC that was disaturated was increased with betamethasone, but decreased with T3. Betamethasone treatment resulted in an increase in the whole lung disaturated PC content, but treatment with T3 did not. Betamethasone administration also increased fetal serum T3 levels, but T3 injection did not produce elevated fetal serum corticosterone levels.Injection of T3 in vivo, or exposure of explants of 18-d fetal lung to 100 nm T3 for up to 48 h did not result in an increase in cytoplasmic glucocorticoid binding or nuclear translocation of the receptor steroid complex. Exposure of explants to glucocorticoid or T3 in vivo or in culture (dexamethasone, 100 nM and T3, 100 nM; for 48 h) resulted in a significant increase in the activity of cholinephosphate cytidylyltransferase, an enzyme in the choline incorporation pathway of PC synthesis. Exposure of explants to the combination of hormones resulted in stimulation that was equal to the sum of that produced by the single hormones but was not statistically significantly different from the glucocorticoid effect. The activities of other enzymes of phospholipid synthesis were not increased by exposure to either hormone, in vivo or in vitro.The additive effects of T3 and glucocorticoid with regard to choline incorporation into PC in fetal rat lung suggest that combined hormone therapy may be useful for the prevention of respiratory distress syndrome in humans. Further animal studies are required, however, before clinical use can be considered.

The influence of hormones on the biochemical development of fetal rat lung in organ culture: I. Estrogen

Abstract It was recently demonstrated that 17β-estradiol enhances phosphatidylcholine synthesis in fetal lung in vivo. In order to determine whether estrogen acts directly on the lung, we examined the influence of 17β-estradiol on phospholipid and glycogen metabolism in explants of 18 day fetal rat lung in organ culture. Exposure of the explants to 17β-estradiol resulted in significant stimulation of [Me- 3 H]choline incorporation into phosphatidylcholine, disaturated phosphatidylcholine and sphingomyelin. Incorporation of [ 3 H]acetate into total phospholipid was increased by 63% ( P P 3 H] acetate in the various phospholipid fractions, it was found that there was a 29% increase in the phos-phatidylglycerol fraction ( P P P These data indicate that estrogen acts directly on the fetal lung and stimulates the incorporation of choline and acetate into phospholipids. It may specifically enhance incorporation into phosphatidylglycerol, the second most abundant phospholipid in pulmonary surfactant.

A null mutation of Hhex results in abnormal cardiac development,defective vasculogenesis and elevated Vegfa levels

The homeobox gene Hhex has recently been shown to be essential for normal liver, thyroid and forebrain development. Hhex–/– mice die by mid-gestation (E14.5) and the cause of their early demise remains unclear. Because Hhex is expressed in the developing blood islands at E7.0 in the endothelium of the developing vasculature and heart at E9.0-9.5, and in the ventral foregut endoderm at E8.5-9.0, it has been postulated to play a critical role in heart and vascular development. We show here, for the first time, that a null mutation of Hhex results in striking abnormalities of cardiac and vascular development which include: (1) defective vasculogenesis, (2) hypoplasia of the right ventricle, (3) overabundant endocardial cushions accompanied by ventricular septal defects, outflow tract abnormalities and atrio-ventricular (AV) valve dysplasia and (4) aberrant development of the compact myocardium. The dramatic enlargement of the endocardial cushions in the absence of Hhex is due to decreased apoptosis and dysregulated epithelial-mesenchymal transformation (EMT). Interestingly, vascular endothelial growth factor A (Vegfa) levels in the hearts of Hhex–/– mice were elevated as much as three-fold between E9.5 and E11.5, and treatment of cultured Hhex–/– AV explants with truncated soluble Vegfa receptor 1, sFlt-1, an inhibitor of Vegf signaling, completely abolished the excessive epithelial-mesenchymal transformation seen in the absence of Hhex. Therefore, Hhex expression in the ventral foregut endoderm and/or the endothelium is necessary for normal cardiovascular development in vivo, and one function of Hhex is to repress Vegfa levels during development.

Glucocorticoid stimulation of choline-phosphate cytidylyltransferase activity in fetal rat lung: receptor-response relationships.

A number of previous studies using in vivo and cultured fetal lung models have shown that the activity of choline-phosphate cytidylyltransferase, the enzyme which catalyzes a rate-limiting reaction in de novo phosphatidylcholine synthesis, is increased by glucocorticoids and other hormones which accelerate fetal lung maturation. To examine the mechanism of this glucocorticoid action further, we examined the effect of dexamethasone on cytidylyltransferase activity in cultured fetal rat lung explants and related it to specific dexamethasone binding. Dexamethasone stimulated cytidylyltransferase activity in the homogenate, microsomal and 105,000 X g supernatant fractions. The hormone did not alter the subcellular distribution of the enzyme, however; the bulk of the activity was in the supernatant fraction in both the control and dexamethasone-treated cultures. The dose-response curves for stimulation of cytidylyltransferase activity in the supernatant fraction and specific nuclear binding of dexamethasone were similar and both plateaued at approx. 20 nM. The EC50 for cytidylyltransferase stimulation was 6.6 nM and the Kd for dexamethasone binding was 6.8 nM. The relative potencies of various steroids for stimulating choline-phosphate cytidylyltransferase and for specific nuclear glucocorticoid binding were the same: dexamethasone greater than cortisol = corticosterone = dihydrocorticosterone greater than progesterone. The stimulation by dexamethasone of cytidylyltransferase activity and of choline incorporation into phosphatidylcholine were both abolished by actinomycin D. These data show that the stimulatory effect of dexamethasone on fetal rat lung choline-phosphate cytidylyltransferase activity is largely on the enzyme in the supernatant fraction and does not involve enzyme translocation to the microsomes as has been reported for cytidylyltransferase activation in some other systems. This effect of dexamethasone is a receptor-mediated process dependent on RNA and protein synthesis.

Delay in Pulmonary Glycogen Degradation in Fetuses of Streptozotocin Diabetic Rats

Summary: The developmental profile of pulmonary glycogen was investigated in fetuses of rats made diabetic before conception with the injection of 40 mg/kg streptozotocin.Lungs of control litters showed increasing pulmonary glycogen concentration from day 16–20, followed by significant decline by term (= 22 days). In contrast, the diabetic litters, which had pulmonary glycogen concentration equal to controls until day 20, showed significantly higher glycogen values (P < 0.01) on days 21 and 22, consistent with a delay in glycogen degradation. This coincided with the finding of decreased amounts of fetal pulmonary phosphatidylcholine and disaturated phosphatidylcholine on day 21 of the diabetic gestation (P < 0.05), but not before that time.Pulmonary glycogen phosphorylase A activity was significantly decreased in the diabetic litters on the final days of gestation, at the same time that the delay in glycogen breakdown became evident. Pulmonary glycogen synthase activity did not differ in the control and diabetic fetuses.Speculation: The delay in pulmonary glycogen degradation seen in the fetus of the diabetic gestation is thus temporally related to the delay in lung maturation seen in this model and may be secondary to a decrease in the activity of the glycogenolytic enzyme phosphorylase A. Decreased availability of pulmonary glycogen stores for surfactant synthesis may be important in elucidating the mechanism of the delayed pulmonic maturation seen in fetuses of diabetic pregnancies.

The influence of hormones on the biochemical development of fetal rat lung in organ culture. II. Insulin.

Summary: The influence of insulin on the biochemical and morphologic maturation of 19-day fetal rat lung (term is 22 days) was studied in an organ culture system. Exposure to insulin for 24 hr resulted in a significant increase in the glycogen content and the rate of glucose oxidation to CO2. In association with this, there was morphologic evidence of delayed lung maturation as evidenced by a significant decrease in the number of lamellar bodies and type II cells in the explants.Insulin treatment had no effect on the rate of choline incorporation into phosphatidylcholine (PC) or disaturated PC and did not antagonize the dexamethasone-induced stimulation of choline incorporation into PC. When the incorporation of [3H]acetate into the various phospholipid fractions was examined, however, it was found that exposure to insulin resulted in a significant decrease in the percentage of phospholipid radioactivity in the disaturated PC fraction and an increase in the percentage of radioactivity in the “membrane” phospholipids, phosphatidylethanolamine, phosphatidylinositol plus phosphatidylserine, and sphingomyelin. There was no significant change in the unsaturated PC and phosphatidylglycerol fractions. Treatment with dexamethasone generally had the opposite effect to insulin with regard to acetate incorporation into the various phospholipid fractions, and when the two hormones were combined, this effect was diminished or abolished. The effects of insulin could not be accounted for on the basis of a change in activity of any of the enzymes of phospholipid synthesis that were examined.These findings suggest that insulin stimulates the synthesis of the cell membrane phospholipids while decreasing that of the surfactant phospholipid, disaturated PC.Speculation: Insulin stimulates lung cell growth while delaying lung cell differentiation.

The influence of postnatal nutritional deprivation on the phospholipid content of developing rat lung.

It has been previously reported that fasting may result in decreased lung surfactant production. In order to investigate this relationship and the role of nutrition in lung phospholipid synthesis, 21-day-old rats were exposed for 60 h to one of five dietary regimens: standard rat chow (controls), fasting, pure glucose, pure fat, or pure protein. After the period of fasting there was a 33% decrease in lung protein content, but there was no change in DNA content. Exposure to any of the experimental diets resulted in a decrease in tissue total phospholipid and phosphatidylcholine content per lung, but not per unit lung protein. Similarly lung lavage phospholipid and phosphatidylcholine content was decreased by 25% after fasting when expressed per lung or per unit DNA, but not per unit protein. Pulmonary cholinephosphotransferase (EC 2.7.8.2) activity was decreased in the fasted animals and those fed the protein diet, but not in the glucose or fat-fed animals. The activities of acetyl-CoA carboxylase (EC 6.4.1.2) and microsomal fatty acid elongation were decreased in all the experimental groups except for the glucose-fed group. It is concluded that fasting results in a decrease in lung cell size but not in lung cell number. Total phospholipid and phosphatidylcholine content in lung tissue and lung lavage is decreased per cell but not per unit cell mass.

Corticosteroid binding by fetal rat and rabbit lung in organ culture

To further characterize glucocorticoid action in fetal lung cells, we investigated corticosteroid metabolism and binding in explants of fetal rat and rabbit lung. Cortisone (E) was concerted to cortisol (F) and bound by receptor with a time course only somewhat slower than for F. Production of F (0.243 pmol/min/mg DNA) was the same in male and female rabbits and was not affected by prior exposure to glucocorticoid in utero or in culture. The t 1/2 for dissociation of nuclear-bound [3H]F was 84 min on changing the culture medium and 21 min on addition of excess non-labeled dexamethasone. Dissociation of [3H]dexamethasone was approx 5-fold slower by both procedures. The KD for nuclear binding of dexamethasone, F, E, and corticosterone in rabbit lung were 0.7, 7.3, 6.8 and 70.6 nM, respectively. In rat lung, the KD for dexamethasone was 6.8 nM. The concentrations of dexamethasone and F required for half-maximal stimulation of phosphatidylcholine synthesis were similar to the KD values. Dexamethasone binding capacity (sites/mg DNA) increased with age in both rat (+103% increase from day 16 to 22) and rabbit (+47% between day 23 and 30). Receptor concentration was the same in both sexes, and there were no developmental changes in non-specific binding, nuclear:cytoplasmic distribution, or KD. In 27-day rabbit fetuses, the rate of choline incorporation was higher in lungs with greater binding capacity. We conclude that (1) E is rapidly converted to F in rabbit lung to become an active glucocorticoid, whereas corticosterone probably has little physiologic activity, (2) there is a species difference in the affinity of dexamethasone binding which is reflected in responsiveness (3) there is no difference between sexes in E conversion, receptor capacity, or phosphatidylcholine synthesis, and (4) the concentration of binding sites per lung cell increases during fetal development. We suggest that developmental increases in both F production and receptor may be important factors in the expression of endogenous glucocorticoid effects.

Insulin antagonism of dexamethasone-induced stimulation of cholinephosphate cytidylyltransferase in fetal rat lung in organ culture

We have studied the antagonism between insulin and dexamethasone on phosphatidylcholine synthesis in fetal rat lung in organ culture. Explants from 18-day fetal rat lungs were cultured in serum-free medium in the presence of 10−6 M dexamethasone phosphate, 1 U/ml bovine insulin or both hormones for 48 h. Control explants were similarly cultured in the absence of hormones. Dexamethasone alone stimulated the activity of cholinephosphate cytidylyltransferase by 134% while insulin alone had no effect. When insulin was added together with dexamethasone, however, the stimulation was reduced to 72%. These data suggest that antagonism by insulin of dexamethasone-induced stimulation of fetal lung phosphatidylcholine biosynthesis may be at least partially expressed at the level of cholinephosphate cytidylyltransferase.