Fuels, hormones, and liver metabolism at term and during the early postnatal period in the rat.

The metabolic response to the first fast experienced by all mammals has been studied in the newborn rat. Levels of fuels and hormones have been compared in the fetal and maternal circulations at term. Then, after cesarean section just before the normal time of birth, sequential changes in the same parameters were quantified during the first 16 h of the neonatal period. No caloric intake was permitted, and the newborns were maintained at 37 degrees C. Activities of three key hepatic enzymes involved in glucose production were estimated. Marked differences in maternal and fetal hormones and fuels were observed. Lower levels of glucose, free fatty acids, and glycerol but higher levels of lactate, alpha-amino nitrogen, alanine, and glutamine were present in the fetus. Pyruvate, glutamate, and ketone bodies were not significantly different. The combination of a strikingly higher fetal immunoreactive insulin and a slightly lower immunoreactive glucagon (pancreatic) resulted in a profound elevation in the insulin-to-glucagon ratio, a finding consistent with an organism in an anabolic state. The rat at birth presents a body composition with respect to fuels available for mobilization and conversion which is dominated by carbohydrate and protein, since little fat is present. However, at birth a transient period of hypoglycemia occurred, associated with a rapid fall in insulin and rise in glucagon, causing reversal of the insulin-to-glucagon relationship toward ratios such as were observed in the mother. After a lag period, hepatic activities of phosphorylase, glucose-6-phosphatase, and phosphoenolpyruvate carboxykinase increased. Concurrent with these enzyme changes, the blood glucose returned to levels at or above those of the fetus. Interestingly, the fall observed in levels of the gluconeogenic precursors, lactate and amino acids, preceded the rise in enzyme activities and restoration of blood glucose. After 4 h, however, hypoglycemia recurred, during a period of decreasing hepatic glycogen content and blood lactate, pyruvate, and glycerol levels but of stable or increasing amino acid concentrations. Hepatic gluconeogenesis in this phase of depleted glycogen stores was insufficient to maintain euglycemia. Substrates derived from fat showed early changes of smaller magnitude. The rise in free fatty acids which occurred was less than twofold the value at birth, though this rise persisted up to 6 h. Whereas glycerol rose transiently, acetoacetate did not change and beta-hydroxybutyrate concentration fell. Both ketone bodies showed a marked rise at 16 h. at a time of diminished free fatty acid levels. Plasma growth hormone, though higher in the fetal than the maternal circulation, showed no consistent change during the period of observation. The changes in levels of the endocrine pancreatic hormones at birth were appropriate in time, magnitude, and direction to be implicated as prime regulators of the metabolic response during the neonatal period in the rat.

Role of alpha-glucosidase in fetal lung maturation.

The role of lysosomal enzyme acid alpha-glucosidase in fetal lung development was investigated with the aid of a specific inhibitor, the pseudosaccharide acarbose. The drug was added to a Waymouth culture medium of fetal rat lung explants cultivated for 48 h from gestational stage 19.5 days, an in vitro system previously shown to allow morphological and biochemical maturation of alveolar epithelium. Glycogenolysis was reduced by 40% as compared with tissue cultivated on control medium, which means that alpha-glucosidase could account for as much as 40% of fetal lung glycogenolysis, the remaining 60% being presumably achieved by cytosolic phosphorylase and by a microsomal neutral alpha-glucosidase. By the same time, the increase of phospholipids of surfactant fraction extracted from cultivated explants was partially inhibited: total and saturated phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were about 30-40% lower than in lungs cultivated on control medium. It should be emphasized that DNA concentration and increases in non-surfactant phospholipids were unchanged by the drug. alpha-Glucosidase activity was evidenced in the lysosomal fraction, in the microsomal fraction and, although in lower amounts, in the surfactant fraction extracted from term fetal lung. The results suggest that lysosomal alpha-glucosidase plays a major role in lung maturation and could facilitate glycogenolysis for the specific use of glycogen stores in providing substrates for surfactant phospholipid biosynthesis.

Maturation of fetal rat lung in diabetic pregnancies of graduated severity.

Biochemical and morphologic maturation of fetal rat lung was studied in pregnant, diabetic rats with different levels of glucose intolerance (sub-, mildly, and severely diabetic). Fetuses were almost normoglycemic and hyperinsulinemic in subdiabetic rats, both hyperglycemic and hyperinsulinemic in mildly diabetic rats, and hyperglycemic but hypoinsulinemic in severely diabetic rats. A similar delay in type II pneumocyte differentiation and a similar decrease of disaturated phosphatidylcholine (DSPC) content in lung tissue and broncho-alveolar material recovered by lung lavage occurred in the fetuses of the three diabetic groups, independently of the severity of diabetes. Phosphatidylglycerol (PG) was decreased in fetuses from severely diabetic rats only. DSPC appeared specifically affected in fetuses of sub- and mildly diabetic groups, whereas in those of severely diabetic groups, DSPC alterations accompanied a variety of abnormalities including whole lung hypoplasia and hypotrophy and decreases of sphingomyelin, unsaturated PC, and lysoPC. The mechanisms leading to abnormal lung development could therefore be different in fetuses of sub- and mildly diabetic rats on one hand and fetuses of severely diabetic rats on the other. Since hyperinsulinemia was the prominent feature of fetal "milieu intérieur" in subdiabetic rats, this study presents arguments gained from in vivo experiments for an implication of hyperinsulinemia in lung developmental retardation due to maternal diabetes. However, the decrease of PG seems to depend on increased blood glucose level in itself. Diminished lung glycogen breakdown and decreased lung triglyceride content, more pronounced in fetuses of sub- and mildly diabetic rats than in those of severely diabetic rats, suggest that in the former, the decrease of DSPC biosynthesis could be due to decreased availability of substrates because of abnormal glycogen utilization. Fetuses from sub- and mildly diabetic rats constitute experimental models most closely resembling the human fetus of the diabetic mother with respect to circulating glucose and insulin. They appear therefore more adequate for elucidating the mechanisms of abnormal lung development in the diabetic pregnancy. In contrast, fetuses from severely diabetic rats associate very high blood glucose levels and hypoinsulinemia, which are features closer to those of adult diabetic subjects than to those of the human fetus of the diabetic mother.

Growth hormone in encephalectomized rat fetuses, with comments on the effects of anesthetics.

Plasma and pituitary immunoreactive growth hormone (GH) was measured in 21.5-day-old rat fetuses under various experimental conditions. Encephalectomy on day 19.5 was used as a method for depriving the fetus of its hypothalamus. The fetuses were recovered on day 21.5 under maternal pentobarbital anesthesia. Total encephalectomy or partial encephalectomy (ablation of superficial brain structures) similarly affected fetal growth. The mean GH contents of the pituitaries were not significantly different in the four groups of fetuses studied: controls from intact females (1.38 +/- 0.19 mug/gland), controls from females submitted to surgery on day 19 (1.47 +/- 0.13 mug/gland), surgically encephalectomized fetuses (1.13 +/- 0.12 mug/gland), sham-operated fetuses (1.19 +/- 0.10 mug/gland). The mean plasma GH levels were the same in control fetuses of intact females (147 +/- 8 ng/ml) and in control fetuses of females submitted to surgery (168 +/- 9 ng/ml). The values were lower in sham-operated fetuses (118 +/- 11 ng/ml) and considerably reduced (P less than 0.001) in encephalectomized fetuses (60 +/- 8 ng/ml). Plasma GH was higher in the fetuses of females killed less than 2 min earlier, than in the fetuses of anesthetized females. In dams anesthetized with pentobarbital or ether, the fetal plasma levels of GH were not different after 15 or 45 min of maternal anesthesia. Under maternal urethane anesthesia, the fetal plasma GH was at 15 min significantly lower than it was under (P less than 0.01) or pentobarbital (P less than 0.05); 30 min later, it had increased by 40% (P less than 0.025). It appears that the release of GH in the fetus can be modified by anesthetics, and that some GH still is released by the pituitary gland in the absence of the hypothalamus.

Somatostatin receptors on rat anterior pituitary membranes.

[125I]Iodo-Tyr1-somatostatin (SRIF) binds with high affinity to one class of sites in the rat anterior pituitary with a KD of 0.91 +/- 0.22 nM and a receptor concentration of 104.4 +/- 1.9 fmol/mg protein. This binding is saturable with respect to tissue concentration and is time-, temperature-, pH-, and calcium-dependent. It is also reversible as a function of time. The rates of association and dissociation were calculated to be 5.98 X 10(7) M-1 min-1 and 0.578 min-1, respectively. Binding of [125I]iodo-Tyr1-SRIF is not inhibited by morphine, beta-endorphin, [D-Ala2]Met-enkephalin, LHRH, TRH, histidylproline diketopiperazine, neurotensin, substance P, bombesin or vasoactive intestinal peptide. In contrast SRIF, [Tyr1]SRIF, and [D-Trp8,D-Cys14]SRIF displace [125I]iodo-Tyr1-SRIF binding with Ki values 0.10 +/- 0.05, 0.46 +/- 0.18, 0.05 +/- 0.01 nM, respectively. The constants of inhibition of a series of alanine monosubstituted analogs of SRIF are correlated (r = 0.89) with their biological potency on GH secretion. Furthermore, postnatal development patterns of [125I]iodo-Tyr1-SRIF binding sites follow the ability of SRIF to inhibit GH release. Thus, [125I]iodo-Tyr1-SRIF binding to adenohypophyseal membranes seems to reflect interaction with SRIF receptors on adenohypophyseal cells. Since biological effects of the peptide have been reported on GH, thyrotropin-stimulating hormone, and PRL secretion, further studies are required to determine the cell types upon which this binding occurs.

Ontogenetic development of the inhibition of growth hormone release by somatostatin in the rat: in-vivo and in-vitro (perifusion) study.

An in-vitro study of GH secretion by rat fetal and neonatal pituitary glands was conducted using a perifusion system. After a 2 h period the GH content of the effluent was constant. Theophylline, thyrotrophin releasing hormone (TRH) and rat stalk median emience extract (SME) were effective stimuli of GH release from the pituitary glands of the 19.5-day-old fetuses. Somatostatin, added to the medium (10 microgram/ml), had no inhibitory effect on GH release (basal or stimulated by either theophylline or SME) before day 4 after birth. After postnatal day 5, somatostatin always inhibited GH secretion. These findings were consistent with the results of experiments in vivo. In rats tested within 4 days of birth, sodium pentobarbitone-stimulated plasma GH levels were not reduced by somatostatin; on day 4 and thereafter somatostatin depressed the response to pentobarbitone injection. These results indicate a postnatal maturation of the regulation of GH release by the hypothalamo-hypophysial system in the rat.

Serum levels of growth hormone during the stunted postnatal development of the vasopressin-deficient Brattleboro rat.

Levels of GH in serum were assayed during the development of heterozygous (HET) control and vasopressin-deficient homozygous (HOM) Brattleboro rats. In early postnatal growth no differences in GH concentrations were present between HET and HOM rats for the rapid decline in serum levels of GH in the first week and the constant period up to day 24 of age thereafter. However, higher values were found in 55-day-old HOM rats and lower values at the age of 9 months. It is concluded that the stunted development of the body and brain of HOM rats is not GH-related, and that changes or anomalies in GH secretion appear only after neurogenesis has been completed.

[Plasma growth hormone in the rabbit fetus. Relation to maturation of the liver and lung]. / Hormone de croissance plasmatique chez le foetus de Lapin. Relations avec la maturation du foie et du poumon.

Day 25 after insemination is a date of peculiar importance in the maturation of several organs in the Rabbit fetus. From day 25 onward the fetal liver stores increasing amounts of glycogen and the lung stores increasing amounts of lecithins, concomitant with sudden rise in the activity of lung phosphatidic-acid phosphohydrolase. Earlier studies on decapitated fetuses established that glycogen storage in the liver is dependent on a dual hormonal control, comprising a pituitary hormone like growth hormone or prolactin (some placental hormones share the same activity) and corticosteroids (Jost, 1961). Since the variations in endogenous corticosteroids do not seem to herald these liver or lung changes (Mulay et al., 1973), a study was made of growth hormone. Plasma immunoreactive growth hormone--determined with a heterologous Rat system (Kervran et al., 1976)--increases eightfold between days 23 and 25. During the same time plasma prolactin does not change according to McNeily and Friesen, 1978, and to unpublished data obtained with Dr McNeilly. In preliminary assays, Rat growth hormone was seen to increase phosphorylase "a" activity in the lung of 18.5 day-old Rat fetuses, thus anticipating normal development. We suggest that growth hormone plays a role in initiating liver and lung maturation.

[Decrease of pulmonary phosphatidylglycerol in the fetuses of rats made diabetic by streptozotocin]. / Diminution du phosphatidylglycérol pulmonaire chez les foetus de Rattes rendues diabétiques par la streptozotocine.

Phosphatidylglycerol and phosphatidylinositol were studied on gestational day 21.5 in the lung of Rat fetuses from streptozotocin-diabetic mothers (severe diabetes; maternal glycemia, 16.5 16.5 mmol/l). A 55% decrease of phosphatidylglycerol and a concomitant 60% increase of phosphatidylinositol were shown. These changes are qualitatively similar to those previously observed in amniotic fluid of human fetuses from diabetic mothers. Since in the severely diabetic pregnant Rat, the fetuses present hyperglycemia and hypoinsulinemia, instead of reactional hyperinsulinemia as the human fetus does, the changes observed in fetal lung are probably due to a direct effect of glucose excess, possibly through an increase of blood myoinositol.

Changes in surfactant phospholipids in fetal rat lungs from normal and diabetic pregnancies.

The purposes of this study were to adapt and evaluate further a pulmonary surfactant isolation method applicable to unperfused fetal rat lung, to quantitate key phospholipids phosphatidylcholine (disaturated phosphatidylcholine, and phosphatidylglycerol) of the isolated material during the last 3 days of gestation, and to determine if abnormalities in surfactant phospholipids were present in fetuses of diabetic pregnancies. A simplified scheme of sucrose gradient centrifugation proved useful for small scale preparations of material enriched in the phospholipids most characteristic of pulmonary surfactant. It was shown that fetal blood phospholipids did not contaminate the surfactant fraction and therefore would not produce artifacts in assessment of lung maturational changes. Analyses of subcellular fractions isolated at 19.5, 20.5, and 21.5 days revealed that the percentages of disaturated phosphatidylcholine relative to total phospholipids were 23-44% in the surfactant preparations and 14-21% in the residual (nonsurfactant) fractions, while the disaturated phosphatidylcholine/phosphatidylcholine ratios were 0.62 +/- 0.06 and 0.41 +/- 0.03, respectively. Summation of the amounts of individual phospholipids in the two fractions yielded data that were nearly identical to the concentrations of these compounds in whole fetal lung samples analyzed independently, implying that losses during the surfactant isolation technique were negligible. The concentrations of phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, and total phospholipids increase markedly (more than 10-fold) and progressively in surfactant fractions prepared from normal fetal rat lung at 19.5, 20.5, and 21.5 days of gestation. In contrast, the residual fractions showed no changes from 19.5 to 20.5 days and then relatively modest increases from 20.5 to 21.5 days, except for phosphatidylglycerol, which increased markedly.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of myo-inositol in impairment of fetal lung phosphatidylglycerol biosynthesis in the diabetic pregnancy: physiological consequences of a phosphatidylglycerol-deficient surfactant in the newborn rat.

To test the hypothesis that the deficit of fetal lung surfactant phosphatidylglycerol (PG) in diabetic pregnancies was due to increased plasma myo-inositol, lung PG content and plasma myo-inositol level were compared in fetuses of streptozotocin-diabetic rats and in fetuses of rats injected with myo-inositol during the 4 last gestational days. An inverse linear correlation was established between circulating myo-inositol and lung phosphatidylglycerol content, including data from fetuses of diabetic rats, which is consistent with the hypothesis. Changes in phospholipid synthetic rates were estimated in fetuses of rats given myo-inositol by measuring incorporation of labelled glycerol on a six hour period on the 21st gestational day, after i.v. injection to the mother. Incorporation into PG was 2.5 times smaller but incorporation into PI or PC were not modified. Pulmonary function in PG-deficient newborns of rats given a high-dosage myo-inositol was assessed by pressure/volume measurements on the lung in situ and by measurement of oxygen tension in aortic blood. Opening pressure of alveoli for lung inflation was increased and blood oxygenation was reduced (30%) in newborns with PG-deficient surfactant as compared with controls, thus suggesting an important physiological role for surfactant PG at birth.

Culture of fetal alveolar epithelial type II cells in serum-free medium.

A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle's minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistence of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.

Plasma levels of growth hormone, corticosterone and insulin, during induced hepatocyte synchronization in young rats.

A wave of synchronous hepatocytes entering the cell cycle can be obtained in vivo after a subcutaneous injection (e.g. of casein) in rats at around Post-natal Day 10, when plasma growth hormone (GH) levels reach a low plateau (40 +/- 2 ng/ml) and liver cell proliferation rate is high. The present work reports the following changes in plasma hormone concentrations after synchronization of 20% of the hepatocyte population: (1) during the G1 phase (i.e. 6-12 hr after the mitogenic trigger), plasma GH concentration has dropped further (25 +/- 1.5 ng/ml). It was back to 90% of control levels during the S phase, mitosis and the following response including a transitory decrease in labelling index below control values. Injected together with the irritating mitotic trigger, a single dose of rat GH reduced the cell synchronization and post-synchronization effects by 50%. (2) Plasma corticosterone levels varied inversely to those of GH, increasing to twice the control values during G1 and were back to physiological levels when synchronized hepatocytes entered the S phase. (3) Variations in insulin levels were similar to that of corticosterone, with narrower ranges and reduced amplitudes. Our data suggest a possible correlation between the observed variations in plasma hormone levels and the induced synchronous hepatocyte response.

Lung maturation in the hyperinsulinemic rat fetus.

Hyperinsulinemic rat fetuses were obtained either by repeated in utero injections of long-acting insulin (resulting in fetal hypoglycemia) or by chronically infusing intravenous glucose to the mother (resulting in fetal hyperglycemia). Fetuses were examined at term. In insulin-injected fetuses (n = 15), surfactant (S) fraction phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) were significantly decreased (3.6 +/- 0.1 nmol Pi/mg tissue; p less than 0.001 and 2.8 +/- 0.1 nmol/mg; p less than 0.025, respectively) as compared with their saline-injected controls (4.8 +/- 0.2 and 3.3 +/- 0.1 nmol/mg, respectively, n = 19). However, residual (R) fraction was unchanged, and there was no difference in whole-lung phospholipids (combined S and R fractions). These results are consistent with morphological data showing a lower lamellar body area per type II cell profile in insulin-injected fetuses as compared with their controls [1.41 +/- 0.13 micron 2 (n = 72) versus 1.99 +/- 0.14 micron 2 (n = 129) p less than 0.01]. Glycogen content was slightly higher in insulin-injected fetuses (18.5 +/- 1.0 micrograms/mg, n = 17) than in their controls (15.1 +/- 0.8 micrograms/mg, n = 18; p less than 0.05). In the second model, changes in S fraction PC and DSPC were similar to those observed after insulin-injections: 4.3 +/- 0.25 and 3.4 +/- 0.2 nmol Pi/mg in fetuses of glucose-infused rats (n = 10) versus 5.7 +/- 0.45 and 4.3 +/- 0.3 nmol Pi/mg, respectively, in controls (n = 10, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Optimization of fetal lung organ culture for surfactant biosynthesis.

Lung organ culture has been a widely used system for studying differentiation and maturation of alveolar epithelium through various culture conditions. The purpose of this work was to carefully characterize in vitro lung biochemical differentiation through isolation of surfactant fraction from tissue and to search for optimal culture conditions. Fetal rat lung was explanted on the 18th gestational day for studying glycogen storage, and on the 20th gestational day for studying surfactant accretion, and cultivated for 48 h. Morphologic differentiation was studied by electron microscopy on tissue explanted on the 17th or 18th gestational days and cultivated for various times. Glycogen storage was greater on fluid medium, although less than occurring in vivo. Cellular integrity and surfactant accumulation were maximal on a semisolid medium containing 0.5% agar. Use of O2-CO2 instead of air-CO2 for gassing the explants slightly decreased phospholipid accumulation. Among media used in previous lung culture studies, Waymouth MB 752/1 was the only one to allow net glycogen accumulation in vitro. The most favorable media for surfactant phospholipid accretion were Waymouth MB 752/1, Eagle's minimum essential and its Dulbecco's modification, CMRL 1066, and NCTC 109. They allowed a 12- to 14-fold increase of surfactant fraction phospholipids in vitro, which is similar to the increase occurring in vivo during the same period. Ham's F10 and F12 media allowed a six fold increase. RPMI 1640 and medium 199 (M199) allowed only a three fold increase. Phospholipid concentration in nonsurfactant fraction only doubled during culture, and differences between various media were much less marked. DNA concentration changed little during culture. Morphologic differentiation of epithelial cells was advanced as compared with in vivo timing in a medium allowing maximal surfactant accretion (Waymouth MB 752/1) but not in a medium allowing low surfactant increase (RPMI 1640). The possible role of compositional differences between media is discussed.

Effects of the antiglucocorticoid RU 486 on the maturation of fetal rat lung surfactant.

The role of endogenous glucocorticoids in the control of surfactant system maturation was investigated in the fetal rat using an antiglucocorticoid molecule synthesized by Roussel-UCLAF, RU 486. The drug was administered to the mother from day 16 of gestation on. In a preliminary step, the transplacental transfer of RU 486 and its antiglucocorticoid effects on fetal target tissues were verified by evidencing RU 486-receptor complexes in fetal liver and lung, by measuring liver glycogen content, and by evaluating fetal blood corticosterone. The maturational state of fetal lungs was assessed biochemically on days 19, 20, and 21 of gestation by measuring their glycogen content, the phospholipid content of whole lung tissue and isolated surfactant fraction, and the incorporation of [methyl-3H]choline into DSPC. Morphological development was studied by analyzing electron micrographs of type II cells. The measured parameters clearly indicated a slowing of maturational processes in lungs of fetuses from RU 486-treated mothers, thereby demonstrating that endogenous glucocorticoids are actually involved in the control of lung maturation. In addition, the obtained results showed that endogenous corticosteroids specifically acted on the surfactant system of the fetal lung.