Effect of starvation on nutrition and insulin secretion in pregnant rats and their fetuses

Pregnancy is thought to create a metabolic condition of accelerated starvation. To clarify this idea, the effect of fasting on pregnant rats (day 21 of gestation) and their fetuses was examined. Although pregnancy significantly increased plasma insulin, plasma ketone body concentrations in fed pregnant rats were higher than those of age-matched fed virgin rats. After 48 hr fasting (i.e., fasting during days 19-21 of gestation), plasma insulin was markedly decreased in virgin rats compared with term pregnant rats, while ketone bodies were significantly higher in pregnant rats than in virgin rats. Body weight was lower in fetuses from fasted mothers than those from fed mothers. Starvation also markedly diminished the insulin response to glucose in isolated, perfused pancreases in both virgin and pregnant rats. The amount of insulin released during glucose stimulation was greater in pregnancy, and the inhibitory effect of 48 hr fasting on insulin release was greater in virgin rats than in pregnant rats. It is possible, therefore, that in term pregnant rats a decrease in insulin release caused by fasting may cause more profound catabolism than in nongravid rats.     

Glucose and ketone bodies production in hepatocytes isolated from fetuses at term--I. Effect of maternal fasting

The effect produced by maternal fasting on glucose and ketone bodies production has been studied in hepatocytes isolated from fetal rat. Maternal fasting produces a decrease in the weight of fetal liver. Maternal fasting produces a decrease in glucose production, both from endogenous substrates and adding lactate (10 mM) to the incubation medium. Maternal fasting produces an increase in ketone bodies production, both from endogenous substrates and adding acetate (5 mM) to the incubation medium.     

The permissive effects of glucocorticoid on hepatic gluconeogenesis. Glucagon stimulation of glucose-suppressed gluconeogenesis and inhibition of 6-phosphofructo-1-kinase in hepatocytes from fasted rats

Production of [14C]glucose from [14C]lactate in the perfused livers of 24-h fasted adrenalectomized rats was not stimulated by 1 nM glucagon but was significantly increased by 10 nM hormone. Crossover analysis of glycolytic intermediates in these livers revealed a significant reduction in glucagon action at site(s) between fructose 6-phosphate and fructose 1,6-bisphosphate as a result of adrenalectomy. Site(s) between pyruvate and P-enolpyruvate was not affected. In isolated hepatocytes, adrenalectomy reduced glucagon response in gluconeogenesis while not affecting glucagon inactivation of pyruvate kinase. A distinct lack of glucagon action on 6-phosphofructo-1-kinase activity was noted in these cells. When hepatocytes were incubated with 30 mM glucose, lactate gluconeogenesis was greatly stimulated by glucagon. A reduction in both sensitivity and responsiveness to the hormone in gluconeogenesis was seen in the adrenalectomized rat. These changes were well correlated with similar impairment in glucagon action on 6-phosphofructo-1-kinase activity and fructose 2,6-bisphosphate content in hepatocytes from adrenalectomized rats incubated with 30 mM glucose. These results suggest that adrenalectomy impaired the gluconeogenic action of glucagon in livers of fasted rats at the level of regulation of 6-phosphofructo-1-kinase and/or fructose 2,6-bisphosphate content.     

Effect of D-galactosamine in vitro on [U-14C]palmitate oxidation, triacylglycerol synthesis and secretion in isolated hepatocytes

Isolated rat hepatocytes were used to study in vitro effects of 10 mM D-galactosamine (GalN) on hepatic fatty acids metabolism. At this concentration, membrane integrity and biochemical competence (i.e., gluconeogenesis and ureogenesis) remained unaffected. Protein synthesis and secretion, as measured by the incorporation of [U-14C]leucine into total and medium protein, was significantly inhibited when incubated for more than 2 h. GalN activated the incorporation of [U-14C]palmitate into triacylglycerols and depressed its utilization in the formation of labelled ketone bodies and 14CO2. Hepatocytes isolated from fasted rats exposed to GalN in vitro did not show any variation in prelabelled triacylglycerol secretion. GalN induced a rapid inhibition of prelabelled triacylglycerol secretion by hepatocytes isolated from fed rats in which this secretion occurred to a larger extent than in hepatocytes isolated from fasted rats. The data reported here suggest that GalN induces a rise of triacylglycerol synthesis by inhibiting the palmitate oxidation pathway and a decrease of triacylglycerol secretion through an early derangement of the secretory pathway.     

The effects of adrenalectomy on the alpha-adrenergic regulation of cytosolic free calcium in hepatocytes

We have previously published that bilateral adrenalectomy in the rat reduces the Ca2+-mediated alpha-adrenergic activation of hepatic glycogenolysis, while it increases the cellular calcium content of hepatocytes. In the experiments presented here, the concentration of cytosolic free calcium (Ca2+i) at rest and in response to epinephrine was measured in aequorin-loaded hepatocytes isolated from sham and adrenalectomized male rats. We found that in adrenalectomized rats the resting Ca2+i was elevated, the rise in Ca2+i evoked by epinephrine was reduced, and the rise in 45Ca efflux that follows such stimulation was depressed. Furthermore, the slope of the relationship between Ca2+i and calcium efflux was decreased 60% in adrenalectomized. Adrenalectomy did not change Ca2+ release from intracellular calcium pools in response to IP3 in saponin-permeabilized hepatocytes. The EC50 for inositol 1,4,5-triphosphate and the maximal Ca2+ released were similar in both sham and adrenalectomized animals. Finally, the liver calmodulin content determined by radioimmunoassay was not significantly different between sham and adrenalectomized rats. These results suggest that 1) adrenalectomy reduces calcium efflux from the hepatocyte, probably by an effect on the plasma membrane (Ca2+-Mg2+)-ATPase-dependent Ca2+ pump and thus alters cellular calcium homeostasis; 2) adrenalectomy decreases the rise in Ca2+i in response to epinephrine; 3) this decreased rise in Ca2+i is not due to defects in the intracellular Ca2+ storage and mobilization processes; and 4) the effects of adrenalectomy on cellular calcium metabolism and on alpha-adrenergic activation of glycogenolysis are not caused by a reduction in soluble calmodulin.     

Effects of adrenalectomy on hormone action on hepatic glucose metabolism. Impaired glucagon activation of glycogen phosphorylase in hepatocytes from adrenalectomized rats.

The effects of adrenalectomy on glucagon activation of liver glycogen phosphorylase and glycogenolysis were studied in isolated hepatocytes. Adrenalectomy resulted in reduced responsiveness of glycogenolysis and phosphorylase to glucagon activation. Stimulation of cAMP accumulation and cAMP-dependent protein kinase activity by glucagon was unaltered in cells from adrenalectomized rats. Adrenalectomy did not alter the proportion of type I and type II protein kinase isozymes in liver, whereas this was changed by fasting. Activation of phosphorylase kinase by glucagon was reduced in hepatocytes from adrenalectomized rats, although the half-maximal effective concentration of glucagon was unchanged. No difference in phosphorylase phosphatase activity between liver cells from control and adrenalectomized rats was detected. Glucagon-activated phosphorylase declined rapidly in hepatocytes from adrenalectomized rats, whereas the time course of cAMP increase in response to glucagon was normal. Addition of glucose (15 mM) rapidly inactivated glucagon-stimulated phosphorylase in both adrenalectomized and control rat hepatocytes. The inactivation by glucose was reversed by increasing glucagon concentration in cells from control rats, but was accelerated in cells from adrenalectomized rats. It is concluded that impaired activation of phosphorylase kinase contributes to the reduced glucagon stimulation of hepatic glycogenolysis in adrenalectomized rats. The possible role of changes in phosphorylase phosphatase is discussed.     

Adrenal glands are essential for activation of glucogenesis during undernutrition in fetal sheep near term

In adults, the adrenal glands are essential for the metabolic response to stress, but little is known about their role in fetal metabolism. This study examined the effects of adrenalectomizing fetal sheep on glucose and oxygen metabolism in utero in fed conditions and after maternal fasting for 48 h near term. Fetal adrenalectomy (AX) had little effect on the rates of glucose and oxygen metabolism by the fetus or uteroplacental tissues in fed conditions. Endogenous glucose production was negligible in both AX and intact, sham-operated fetuses in fed conditions. Maternal fasting reduced fetal glucose levels and umbilical glucose uptake in both groups of fetuses to a similar extent but activated glucose production only in the intact fetuses. The lack of fasting-induced glucogenesis in AX fetuses was accompanied by falls in fetal glucose utilization and oxygen consumption not seen in intact controls. The circulating concentrations of cortisol and total catecholamines, and the hepatic glycogen content and activities of key gluconeogenic enzymes, were also less in AX than intact fetuses in fasted animals. Insulin concentrations were also lower in AX than intact fetuses in both nutritional states. Maternal glucose utilization and its distribution between the fetal, uteroplacental, and nonuterine maternal tissues were unaffected by fetal AX in both nutritional states. Ovine fetal adrenal glands, therefore, have little effect on basal rates of fetal glucose and oxygen metabolism but are essential for activating fetal glucogenesis in response to maternal fasting. They may also be involved in regulating insulin sensitivity in utero.     

Effects of endogenous and exogenous glucocorticoids on liver differentiation

The effects of maternal bilateral adrenalectomy on day 1 of gestation and betamethasone treatment on fetal liver development were compared, in terms of biochemical and morphological parameters. For fetuses 20 days old (E20), absence of maternal glucocorticoids during gestation caused an increase in the number of nuclei in whole livers, and a significantly decrease of both body weight and protein content per nucleus, in comparison with the control group (C). Betamethasone injection on days 15, 16 and 17 of gestation into adrenalectomized pregnant rats (ADX + BET) did not completely prevent these effects. The electron microscopic analysis of the ADX fetal liver (E20) showed some hepatocyte lesions such as loss of cytoplasmic organelles, increase in hematopoietic cell number as well as a lower cellular maturation in comparison with the control group. The fetal liver from ADX + BET mothers 20 days after gestation displayed a noticeable involution of the hematopoietic component in spite of its relatively immature stage. However, there was no significant change in the degree of fetal hepatocyte lesions. Therefore, supply of maternal glucocorticoids from the beginning of gestation is essential for maintenance of the integral structure of the rat fetal hepatic parenchyma, for the correct maturation of the blood strains and for the beginning of involution of the hematopoietic tissue at the end of gestation.     

IDENTIFICATION OF THE ADRENOCORTICOTROPHIN-PRODUCING CELLS IN THE RAT HYPOPHYSIS BY AUTORADIOGRAPHY

The relative rates of protein (hormone) synthesis and secretion by the various cell types in the anterior hypophysis of the rat have been studied by means of autoradiography. Normal and adrenalectomized male rats were injected with tritiated glycine and their hypophyses removed and fixed at 20, 40, and 90 minutes and 15 hours after injection. Autoradiograms of the hypophysial sections were prepared and autoradiographic grains were counted in the film overlying the cytoplasm of individual cells. With the aid of this method, a unique cell type was identified in the hypophyses of adrenalectomized rats. This cell is morphologically distinct from "gonadectomy cells," "thyroidectomy cells," and from previously described normal cell types, and is therefore designated as the "adrenalectomy cell." Among the 7 cell types differentiated in this study, the "adrenalectomy cell" had the highest tritium content and, in addition, at the time intervals studied this cell had the fastest rate of appearance and disappearance of protein tritium. This autoradiographic evidence of rapid protein (or polypeptide) turnover following adrenalectomy indicates that the "adrenalectomy cell" is the site of adrenocorticotrophin production in the adrenalectomized rat. Further autoradiographic and cytological evidence is presented which suggests that the "adrenalectomy cells" may be derived from chromophobes, and that a portion of the "large chromophobes" as defined in this study may be the site of adrenocorticotrophin production in the normal rat.     

Effect of sodium fluoride on cyclic AMP production in rat hepatocytes.

The effect of NaF on cAMP production was studied in hepatocytes isolated from fed and fasted rats. A four-six fold increase in hepatocyte cAMP production was observed in the presence of 10-20 mM NaF in cells isolated from either fed or fasted rats. The maximal stimulation of cAMP production was observed after a 10 min incubation in the presence of 1 mM theophylline. However, as little as 0.05-0.15 mM NaF induced a significant increase in cAMP production. It was also found that NaF would alter the production of glucose in isolated rat hepatocytes. When hepatocytes from fed rats were incubated with 0.05-5 mM NaF there was an increase in amount of glucose released from endogenous sources. Also NaF resulted in a decrease in lactate and pyruvate production. Similarly NaF stimulated glucose production in hepatocytes from fasted rats. The maximal stimulation was observed with about 0.15-0.25 mM NaF. At NaF concentrations greater than 1.5 mM a decrease in glucose production was observed. It is concluded that NaF increases the level of cAMP and alters glucose metabolism in intact hepatocytes.     

Metallothionein synthesis in foetal, neonatal and maternal rat liver

The synthesis of hepatic metallothionein relative to other cytosol proteins was measured by [35S]cysteine incorporation in foetal, neonatal and pregnant rats. The relative rate of hepatic metallothionein synthesis reached a maximum in foetal liver on days 18-21 of gestation. Metallothionein synthesis then declined until weaning, when adult levels were established. The rate of metallothionein synthesis was greater in pregnant rats at term than in nulliparous rats. To determine if circulating inducing agents could play a role in the regulation of metallothionein synthesis in foetal liver we treated pregnant rats with inducers at a time prior to the normal rise in foetal liver metallothionein synthesis. Injections of copper, cadmium or hydrocortisone to 17-day-pregnant dams failed to induce foetal metallothionein synthesis. In contrast, zinc injection to the dam was an effective inducer in the foetuses. Maternal laparotomy (performed to expose the foetus for direct injection of inducers) induced foetal metallothionein synthesis. Metallothionein synthesis in the livers of 17-day-gestation dams was induced by all metal injections and laparotomy but, surprisingly, not by hydrocortisone injection. Maternal adrenalectomy did not influence the subsequent normal elevation in foetal or maternal metallothionein synthesis. These results, in conjunction with previous reports, suggest that mobilization of zinc in serum during late gestation may regulate foetal and maternal changes in metallothionein synthesis.     

Changes in the ultrastructure of rat hepatocyte nucleoli during bilateral adrenalectomy and after administration of cortisol

Nucleolar ultrastructure of the rat hepatocytes after bilateral adrenalectomy and cortisol stimulation has been studied by the electron-microscopic method Traits of nucleolar inactivation (a decrease of granular component enlargement of fibrillar centres, condensation of peri- and intranucleolar chromatin, etc.) are observed in hepatocyte nucleoli 5 days after adrenalectomy. Cortisol stimulation of hepatocytes of the adrenalectomized rats leads to nucleolar activation (4h, 5h, 8h after the hormone injection). Adrenalectomy with following cortisol injection is a useful model to study inactivation and activation of ribosomal genes in the target cells.     

Nutritional control of respiratory and other muscular activities in relation to plasma prostaglandin E in the fetal sheep

The effects of nutrient availability on fetal plasma prostaglandin E (PGE) concentrations, on fetal breathing movements and electromyographic (EMG) activities of fetal nuchal and forelimb muscles were investigated in pregnant ewes by varying dietary intake and by manipulation of fetal plasma glucose concentration. The incidence of fetal breathing movements (06.00-10.00 h) decreased with increasing gestational age while fetal arterial concentrations of plasma PGE increased significantly over the same period of gestation. Maternal fasting for 48 h reduced the incidence of fetal breathing movements and the amount of nuchal EMG activity (06.00-10.00 h) in animals older than 130 days but had no effect earlier in gestation. No changes in forelimb EMG activity were observed during fasting at any gestational age. Plasma PGE levels increased significantly during fasts begun both before and after 130 days of gestation. When data from fed and fasted states were combined for all fetuses, irrespective of gestational age, there was a significant inverse correlation between fetal breathing movements incidence and plasma PGE concentration in utero. This relationship was even more pronounced when the fetuses were considered individually. Insulin infusions induced hypoglycaemia, an increase in fetal plasma PGE concentration and a significant reduction in the incidence of fetal breathing movements at all ages. Glucose infusions of fetal breathing movements only after 130 days and had no effect on plasma PGE levels in utero at any gestational age. Neither insulin nor glucose infusions altered the EMG activities of the nuchal and forelimb muscles. The results show that glucose availability is an important factor in determining the incidence of fetal breathing movements in utero and indicate that nutritionally induced changes in fetal breathing movements are mediated in part by PGE. They also suggest that PGE is a physiological regulator of fetal breathing movements in the sheep during late gestation.     

Ketone body metabolism in the mother and fetus

Pregnancy is characterized by a rapid accumulation of lipid stores during the first half of gestation and a utilization of these stores during the latter half of gestation. Lipogenesis results from dietary intake, an exaggerated insulin response, and an intensified inhibition of glucagon release. Increasing levels of placental lactogen and a heightened response of adipose tissue to additional lipolytic hormones balance lipogenesis in the fed state. Maternal starvation in late gestation lowers insulin, and lipolysis supervenes. The continued glucose drain by the conceptus aids in converting the maternal liver to a ketogenic organ, and ketone bodies produced from incoming fatty acids are not only utilized by the mother but cross the placenta where they are utilized in several ways by the fetus: as a fuel in lieu of glucose; as an inhibitor of glucose and lactate oxidation with sparing of glucose for biosynthetic disposition; and for inhibition of branched-chain ketoacid oxidation, thereby maximizing formation of their parent amino acids. Ketone bodies are widely incorporated into several classes of lipids including structural lipids as well as lipids for energy stores in fetal tissues, and may inhibit protein catabolism. Finally, it has recently been shown that ketone bodies inhibit the de novo biosynthesis of pyrimidines in fetal rat brain slices. Thus during maternal starvation ketone bodies may maximize chances for survival both in utero and during neonatal life by restraining cell replication and sustaining protein and lipid stores in fetal tissues.     

Relationships among surfactant fraction lipids, proteins and biophysical properties in the developing rat lung

Lung development is associated with increases in specific phospholipids and proteins that function as critical pulmonary surfactant components. Attempts to characterize the pattern of surfactant development in fetal rat lungs have been hampered by the lack of a micromethod which will permit quantitative isolation of surface active components from small tissue specimens. As part of studies designed to elucidate the metabolic regulation of lung development in the rat, we developed sucrose density gradient centrifugation procedures to separate pulmonary phospholipids and proteins into a presumed surfactant (S) fraction and a residual (R) fraction. Electron microscopy of S pellets from mature fetuses identified predominant lamellar bodies and minimal contamination; incubation with 5 mM CaCl2 induced the appearance of tubular myelin figures, implying functional potential. This was confirmed by demonstrating low surface tension (less than 1 dyn/cm) in S, but not R, fractions at term gestation (21.5 days) and in 1-day-old neonatal lung isolates, based on dynamic measurements using the oscillating bubble technique. Surface activity was also high in the S pellets from fetuses at 20.5 days of gestation; however, at 19.5 days, minimum surface tension values of at least 19 dyne/cm were seen. These results correlated directly with biochemical analyses which indicated striking increases in three surfactant-associated proteins (SP-A, SP-B, and SP-C) after 19.5 days of gestation; a finding in agreement with previously reported data on the developmental increase of disaturated phosphatidylcholine in fetal rat lung. We conclude that isolation of S fraction components is valuable for demonstrating maturation of the fetal rat lung and may provide a useful tool for the study of regulatory mechanisms influencing surfactant production and function.     

Conversion of pyruvate into ketone bodies in rat hepatocyte suspensions.

The contribution of pyruvate to ketogenesis was determined in rat hepatocyte suspensions by using [14C]pyruvate. The rates of conversion of pyruvate into ketone bodies in hepatocytes from fed and 24 h-starved rats were 10 and 17 mumol/h per g wet wt. respectively, and accounted for 50 and 29% of the total ketone bodies formed. In hepatocytes from fed rats, the addition of palmitate (0.25-1 mM) increased the rate of conversion of pyruvate into ketone bodies (80-140%), but decreased the relative contribution of pyruvate to total ketogenesis. In hepatocytes from starved rats, palmitate did not increase pyruvate conversion into ketone bodies.     

Induction of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes. Requirement for insulin and dexamethasone

To determine the relative contributions of glucose, insulin, dexamethasone, and triiodothyronine to the induction of hepatic glucose-6-phosphate dehydrogenase, hepatocytes isolated from normal or adrenalectomized rats, either fasted or fed, were examined in culture. Addition of insulin (42 milliunits/ml, 0.9 microM) and dexamethasone (1 microM) to hepatocytes obtained from 3-day-fasted rats and cultured for 48 h in serum-free Dulbecco's medium resulted in a 7- to 11-fold increase in Glc-6-P dehydrogenase specific activity compared with a 2- to 3-fold increase in activity in control cultures incubated without added hormones. The effects of insulin and dexamethasone were independent of DNA synthesis, dose-dependent, and additive; each contributing about one-half of the total response. Medium glucose was neither sufficient nor necessary for the insulin- or dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Addition of triiodothyronine (10 microM) preferentially blocked the dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Insulin failed to stimulate the induction of Glc-6-P dehydrogenase in hepatocytes obtained from normal fed rats or from fasted and fed adrenalectomized rats. However, insulin caused a significant increase in the Glc-6-P dehydrogenase specific activity of these cells when dexamethasone was concurrently added to the culture medium.     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.     

Effects of glucocorticoids and catecholamines on maturing rat heart

1. The negative force-frequency response of normal rat heart was accentuated when the animals were adrenalectomized. Treatment of adrenalectomized animals with dexamethasone restored the normal force-frequency response. 2. Total adrenalectomy increased the sensitivity of rat heart to calcium. 3. Adrenalectomized-dexamethasone-treated hearts were more responsive to epinephrine and ouabain. 4. Total adrenalectomy caused independent myocardial disturbances in calcium handling elements (glucocorticoid effect) and beta receptors (catecholamine effect).