Postnatal Rat Liver Research Articles

Glycogenolytic effects of the calcium ionophore A23187, but not of vasopressin or angiotensin, in foetal-rat hepatocytes.

Vasopressin, angiotensin and phenylephrine stimulate glycogenolysis in postnatal rat liver by a Ca2+-mediated mechanism not involving cyclic AMP. To determine whether these hormones promote glycogenolysis in foetal liver, we have examined their effects, and those of the Ca2+ ionophore A23187, on glycogen metabolism in cultured foetal-rat hepatocytes. Vasopressin and angiotensin (0.1 nM-0.1 microM) had no effects on either glycogen synthesis (as assessed by [14C]glucose incorporation into glycogen) or phosphorylase a activity. However, A23187 at 1 and 10 microM inhibited glycogen synthesis by 31.3 and 89.1% respectively (both P less than 0.001) and stimulated phosphorylase a activity by 66.9 and 184.1% respectively (both P less than 0.01). Incubation of cells in Ca2+-deficient medium attenuated the effects of 10 microM-A23187 on glycogen synthesis and abolished the effects of 1 microM-A23187. As in postnatal liver, glucagon (1 and 20 nM) and isoprenaline (1 and 10 microM), which activate adenylate cyclase, inhibited glycogen synthesis and stimulated phosphorylase a activity in foetal hepatocytes. The minimal effective concentration of phenylephrine was 10 times that of isoprenaline. These results indicate striking differences in the ontogeny of cyclic AMP-mediated and Ca2+-mediated processes which regulate hepatic glycogenolysis. Since increases in cytosolic Ca2+ induce glycogenolysis in foetal-rat liver, the weak or absent responses to vasopressin, angiotensin and the alpha-adrenergic agonists may result from defects in hormone-receptor binding or in post-receptor events leading to the mobilization of intracellular Ca2+ stores.

GLYCOGENOLYTIC EFFECTS OF THE CALCIUM IONOPHORE A23187, BUT NOT VASOPRESSIN OR ANGIOTENSIN, IN FERTALRAT HEPATOCYTES

Vasopressin, angiotensin II and phenylephrine stimulate glycogenolysis in postnatal rat liver by a non-cAMP, calcium-mediated mechanism. To determine whether these hormones also promote glycogenolysis in fetal liver, we have examined their effects, and those of the calcium ionophore A23187 (A), on glycogen metabolism in cultured fetal rat hepatocytes. Vasopressin and angiotensin (10−10-10−7M) had no effects on either 14C-glucose incorporation into glycogen or phosphorylase a activity. However, A at concentrations of 1 and 10 uM inhibited glycogen synthesis by 31.3 and 89.1 percent, respectively (p< 0.001) and stimulated phosphorylase a activity by 66.9 and 184.1 percent respectively (p < 0.01). Incubation of cells in calcium-deficient medium markedly attenuated the effects of A on glycogen synthesis. As in postnatal liver, glucagon (1 and 20 nM) and isoproterenol (1 and 10 uM), which activate adenylate cyclase, inhibited glycogen synthesis and stimulated phosphorylase a activity in fetal hepatocytes. The minimal effective concentration of phenylephrine was 10 times that of isoproterenol. These results indicate striking differences in the ontogeny of cAMP-mediated and non-cAMP, calcium-mediated processes which regulate hepatic glycogenolysis. Since increases in cytosolic calcium induce glycogenolysis in fetal rat liver, the weak or absent responses to vasopressin, angiotensin and α - adrenergic agonists may result from defects in hormone-receptor binding or in postreceptor events leading to the mobilization of intracellular calcium stores. NIH grants HD07447 and HDO6301.

Effect of chenodeoxycholic acid feeding during gestation in the rat on bile acid metabolism and liver morphology.

Chenodeoxycholic acid (CDCA) was fed to pregnant rats at the 0.25% level in the diet from Day 11 of gestation to delivery in order to evaluate the effects on (1) maternal tissue bile acid composition, (2) neonatal tissue bile acid composition and cholesterol-7 alpha-hydroxylase activity, and (3) maternal, neonatal, and postnatal liver morphology. Feeding CDCA increased maternal lithocholic acid while significantly decreasing deoxycholic acid, cholic acid, and total bile acids. Feeding CDCA resulted in a significantly higher chenodeoxycholic acid pool in the neonates while neonatal plasma cholesterol and the 7 alpha-hydroxylation of cholesterol was not significantly affected. Morphological examination of maternal, neonatal, and postnatal rat liver revealed no significant hepatotoxicity. This investigation has shown that (a) neonates of CDCA fed dams have a significantly greater pool of CDCA, suggesting maternal-to-fetal transfer of dihydroxy bile acids, (b) neonatal cholesterol-7 alpha-hydroxylase activity and total tissue bile acid pools are not significantly altered by increased pool of CDCA, and (c) no hepatotoxic effects on maternal, neonatal, and postnatal livers were evident with gestational feeding of CDCA at the 0.25% level in the rat.

Effect of maternal ethanol ingestion during pregnancy and lactation on the structure and function of the postnatal rat liver plasma membrane: Assessment with [ 3H]prazosin binding to the hepatic α1-adrenergic receptors

A liquid low-fat nutritionally adequate Metrecal diet in which alcohol contributed 37% of the total calories was given to pregnant rats and maintained during lactation. Control rats were pairfed with an isocaloric sucrose-Metrecal diet. After birth, litters were killed at different ages (days 1–30), and the results showed that growth and survival of progeny from the alcohol-treated rats were adversely affected. Likewise, the wet weights of livers from such pups were consistently less than from the pair-fed controls. The yield of hepatic plasma membrane protein per wet liver weight was constant and independent of either age or diet. Using [ 3H]prazosin as radioligand, equilibrium binding studies were carried out to monitor changes in the structure and function of the plasma membrane in the new-born pups concomitant with the development of α 1-adrenergic receptors. Results obtained with the alcohol-fed pups showed that the binding affinity ( K D ) was not altered throughout. However, the receptor density ( B max) was decreased significantly. This decrease ranged from 60 to 70% in pups 6- to 15-days-old; 45% at 20 days; and 30% in pups at 25 and 30 days of age. These observations suggest that maternal ethanol ingestion affected the posrnatal development of rat liver plasma membranes. Furthermore, by using the hepatic α 1-adrenergic receptor as a metabolic probe, we deduce that a possible impairment exists in the capacity of the alcoholic progeny to respond to the hormonal action of epinephrine. Such a defect may contribute to impaired growth and metabolism in these young animals.

An organ culture of postnatal rat liver slices.

A technique for the organ culture of postnatal and adult rat liver has been developed. Liver slices, 0.3 mm thick, were maintained in Conway units at the interphase between medium and a 95% O2:5% CO2 atmosphere. Postnatal liver in culture for up to 72 h had healthy hepatocytes throughout the explants; if adult liver was used the upper 0.2 mm was healthy after 24 h. These slices incorporated tritiated orotate and leucine into trichloroacetic acid-precipitable material. Incorporation of orotate was shown to be spread over the entire slice of neonatal liver. Culturing did not alter the potassium ion content of postnatal liver. Tyrosine aminotransferase activity in liver slices from postnatal, adult, and adrenalectomized adult rats was stimulated by glucocorticoids and dibutyryl cyclic AMP. Cycloheximide and actinomycin D prevented this response. Further, cortisol exerted a permissive effect on the stimulation of tyrosine aminotransferase activity by dibutyryl cyclic AMP in slices from adrenalectomized rats. Induction of urea cycle enzymes by cortisol was demonstrated in cultures of liver from adrenalectomized adult animals.

Perinatal changes of fructose 2,6-bisphosphate in the rat liver

In order to investigate a possible regulatory role of fructose 2,6-bisphosphate in early developmental stages, where profound changes in the carbohydrate metabolism are known to occur, this effector was estimated in fetal and postnatal rat liver. Polyphasic changes of the hepatic fructose 2,6-bisphosphate levels were found, which could be correlated to alterations in the glucose metabolism. A minimum in the hepatic fructose 2,6-bisphosphate level at the −3rd day coincides with the initiation of glycogen synthesis and its increase two hours after birth concurs with glycogen mobilization.

Comparative effects of insulin and hydrocortisone on glycogen content of fetal and newborn rat liver cultures

Organ cultures prepared from 18- to 19-day fetal and 3- to 6-day-old newborn rat liver were maintained for 2 days in Trowell's T8 medium without insulin and supplemented with 0.1% albumin and 300 mg% glucose. The atmosphere for culture was 95% O 2 and 5% CO 2. Medium alone was used for control cultures, whereas insulin, hydrocortisone, or insulin plus hydrocortisone were used in experimental groups. Explant glycogen stores were maintained better in cultures grown in hormone-supplemented media than in control cultures. Fetal explants were found to have higher levels of glycogen than controls in the insulin or insulin plus hydrocortisone groups. Postnatal explants did not have higher levels of glycogen in the hormone-treated groups. Gestational age appeared to determine whether the liver explants reacted to hydrocortisone or insulin to maintain glycogen stores. Enzymatic assays, in vitro of glycogen synthetase and phosphorylase, indicated that the fetal liver response to insulin plus hydrocortisone by increasing the total and independent form of glycogen synthetase; but similar enzyme studies on postnatal rat liver did not show convincing differences as to an effect on synthetase. No definite in vitro temporal relationships could be identified. Late in gestation, the effect of hydrocortisone on glycogen synthesis is apparently dependent on the presence of insulin. Insulin appears to be required for glycogen storage in vitro in the cultures of postnatal rat liver.

Selective changes in microsomal enzymes of triacylglycerol and phosphatidylcholine synthesis in fetal and postnatal rat liver. Induction of microsomal sn-glycerol 3-phosphate and dihydroxyacetonephosphate acyltransferase activities.

Selective changes in microsomal enzymes of triacylglycerol and phosphatidylcholine synthesis in fetal and postnatal rat liver. Induction of microsomal sn-glycerol 3-phosphate and dihydroxyacetonephosphate acyltransferase activities.

The inducibility of tyrosine aminotransferase in monolayer cultures of hepatocytes from neonatal rats.

Hepatocytes from neo- and postnatal rat liver were isolated, purified from non-hepatocytes (erythropoietic cells), and cultured in sufficient quantity to investigate enzyme inducibility. Tyrosine aminotransferase (TAT) in neo- and postnatal hepatocytes was induced by maximally responsive doses of glucagon, dexamethasone, DB-cAMP, theophylline, and combinations thereof. In cultures from newborn parenchymal cells TAT enzyme-specific activity showed only a moderate inducibility; however, responsiveness to the combination was fully developed 10 days after birth and did not differ from values found in adult liver cells. The results also show the existence of the "permissive" effect of glucocorticoid during postnatal age, and indicate that the development of a possibly involved receptor complex for the induction of TAT is largely completed 5-10 days after birth.

Amino acid transport and rubidium-ion uptake in monolayer cultures of hepatocytes from neonatal rats.

Amino acid and K(+) transport during development has been investigated in hepatocyte monolayer cultures with either alpha-amino[1-(14)C]isobutyrate or (86)Rb(+) used as a tracer for K(+). Parenchymal cells from neo- and post-natal rat livers have been isolated by an improved non-perfusion technique [Bellemann, Gebhardt & Mecke (1977)Anal.Biochem.81, 408-415], and the resulting hepatocyte suspensions purified from non-hepatocytes before inoculation. In the presence of Na(+) (Na(+)-dependent component), the rates of amino acid uptake in neonatal hepatocytes were markedly enhanced compared with cells from 30-day-old rats. When Na(+) was replaced by choline (Na(+)-independent component) the accumulation of alpha-aminoisobutyrate was decreased and it was not affected by the age of the animals. Kinetic analysis of Na(+)-dependent alpha-aminoisobutyrate transport revealed the existence of a high-affinity low-K(m) component (K(m)0.91mm) with a V(max.) of 2.44nmol/mg of protein per 4min, which later declined gradually with progressive development. Rates of Rb(+) transport were concomitantly enhanced in neonatal hepatocytes and thereafter declined with postnatal age. The increased Rb(+) influx was effectively inhibited by ouabain and reflected elevated activity of the electrogenic Na(+)/K(+)-pump during early stages of development. Kinetic evaluation of the enhanced rates of Rb(+) uptake indicates multiple and co-operative binding sites of the enzyme involved in the Rb(+) uptake, and the transport system is positively co-operative (the Hill coefficient h is >1.0). In short, amino acid transport in neonatal rat hepatocytes is increased as a result of an existing low-K(m) component for the Na(+)-dependent alpha-aminoisobutyrate uptake, which endows the hepatocytes with a high capability for concentrating amino acids at low ambient values. The concomitant enhancement of K(+) transport reflects changes in the electrochemical gradient for Na(+) across the hepatocellular membrane and, along with this, presumably alterations in the membrane potential; the latter might be the driving force for the enhanced alpha-aminoisobutyrate transport in the alanine-preferring system during postnatal age.

Synthesis of threonine dehydratase in neonatal rat liver.

Antiserum against rat threonine dehydratase has been raised in rabbits. The rate of incorporation of [3H]leucine into threonine dehydratase was found to increase dramatically after birth. Actinomycin D suppressed the rate of the synthesis of the enzyme in the postnatal rat liver, although the antibiotic was without effect on the incorporation of leucine into total cytosolic protein. The administration of dibutyryl-adenosine 3',5'-monophosphate to foetal rats in utero or postnatal rats resulted in a marked enhancement of the rates of synthesis of threonine dehydratase.

Preparation of primary monolayer cultures of postnatal rat liver cells

We describe a perfusion with collagenase followed by dissociation with collagenase to isolate hepatocytes from 7 to 10-d postnatal rat liver. Subsequent culture in an arginine-deficient medium selects for hepatocytes by preventing overgrowth by other cell types that are unable to synthesize arginine.

Comparison of cytochrome P-450 levels in adult rat liver, postnatal rat liver, and primary cultures of postnatal rat hepatocytes

A system of primary cultures of postnatal rat hepatocytes has been developed to serve as an experimental model for drug metabolism and toxicity investigations. The purpose of this study was to examine the reported loss of cytochrome P-450 of hepatocytes when placed in culture and to compare activity in culture to intact liver and freshly isolated hepatocytes. A medium enriched with several hormones and a system of floating filters as a substratum for cell attachment were investigated as methods to reduce the expected loss of cytochrome P-450. When compared to initial values of cytochrome P-450 in whole liver and isolated hepatocytes, these methods failed to prevent the reduction of cytochrome P-450 in culture. However, our results compare favorably with other values reported in the literature.

A centrifugation study of rat-liver mitochondria, lysosomes and peroxisomes during the perinatal period.

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose-6-phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5'-Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme was used, does not appreciably differ for the late foetal, early postnatal and adult rat livers. An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distribution of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal; foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.

The synthesis of glycosaminoglycans in aging rat liver. A brief note

The synthesis of glycosaminoglycans (GAG) was studied in liver slices from postnatal (9 days), young (140 days), adult (490 days) and senescent (940 days) rats. It was found that the rate of synthesis was highest in postnatal rat liver and decreased to about half in young rats with no further reduction in adult and senescent age groups. The specific radioactivity of the precursors of GAG synthesis did not change with age. The synthesis pattern of specific types of GAG in postnatal liver was characterized by a significant higher percentage of chondroitin sulfate and hyaluronic acid. In the following age classes the profile of specific GAG synthesis did not change significantly (heparin sulfate: chondroitin sulfate: hyaluronic acid: “ keratin sulfate” = 84%:8.3%:1.5%:1.6% ).

Primary monolayer cultures of postnatal rat liver cells with extended differentiated functions.

Monolayers of liver cells cultured from postnatal rats were grown in two types of media. One set of cultures was grown in selective medium which contained ornithine but was deficient in arginine; the other set was grown in nonselective medium which contained arginine but no ornithine. The cultures that were grown in the nonselective medium contained primarily a mixture of two cell types found in the liver: parenchymal hepatocytes and fibroblast-like cells. The fibroblast cells tended to overgrow the hepatocytes after several days in culture. In contrast, fibroblast overgrowth was inhibited in cultures grown in the selective, arginine-deficient medium, thereby resulting in relatively pure cultures of functional parenchymal hepatocytes. Comparative studies of sulfobromophthalein (BSP) uptake showed that the cultures grown in selective medium continued to be active much longer than the cultures grown in the nonselective medium. Pyruvate kinase assays revealed that the cultures grown in selective medium contained primarily the L-isoenzyme type which is characteristic of parenchymal hepatocytes. Cultures grown in nonselective medium contained a mixture of L- and M-isoenzymes which is indicative of nonparenchymal liver cells. The reported results indicate that selective, arginine-deficient medium permits primarily the growth of parenchymal hepatocytes found in neonatal rat liver.

Organ culture of postnatal rat liver: Effects of ethanol on lipid metabolism

Organ culture explants of 3- to 5-day-old postnatal rat liver were used to examine in vitro hepatocyte lipid metabolism as affected by incubation with 1.0% ethanol, v/v, 173 m M) for 6 hr. Lipogenesis, as measured by [ 14C]acetate incorporation into explant lipids, and fatty acid esterification, measured by [ 14C]palmitate incorporation into triglycerides, were both increased by ethanol administration. Production of 14CO 2 by the explants from [ 14C]palmitate, [ 14C]acetate, and [ 14C]glucose was decreased following incubation with ethanol. Ethanol did not affect the release of labeled lipids from the explants into the media. Increases in the explant lactate/pyruvate ratio during incubation with ethanol and incorporation of [ 14C]ethanol into fatty acids indicated that the explants were oxidizing ethanol to acetate. Explant viability, as judged by [ 3H]leucine incorporation into cellular protein and morphologic appearance by light and electron microscopy, was unaffected by incubation with 1.0% ethanol for up to 24 hr. Despite apparently increased lipogenesis and decreased fatty acid oxidation by the explants during ethanol administration, no statistically significant ethanol-induced increases in explant triglyceride or fatty acid concentration were found unless the explants were incubated with media supplemented to 25% serum as an exogenous lipid source. These results indicate that acute hepatic explant lipid accumulation during ethanol administration is due primarily to deposition of exogenous lipids, with increased lipogenesis contributing only minimally to the increased deposition of lipids.

Hormonal induction of threonine dehydratase in postnatal rat liver

1. 1. Insulin administration to neonatal rats results in either potentiation or repression of the development of hepatic threonine dehydratase according to the dosage employed. 2. 2. Simultaneous injections of glucagon and adrenaline to newborn rats do not elicit a greater inductive effect than administration of each hormone alone. 3. 3. No additivity of the inductive effect of glucagon and cyclic AMP can be demonstrated. 4. 4.|Density gradient ultracentrifugation and polyacrylamide gel electrophoresis suggest that the enzymes induced by glucagon, adrenaline and cyclic AMP are identical.

Cytoplasmic binding of dexamethasone and induction of tyrosine aminotransferase in neonatal rat liver

Dexamethasone administration markedly increases the activity of tyrosine aminotransferase in postnatal rat liver. The glucocorticoid fails to induce the enzyme in foetal rats when administered in utero. Dexamethasone binding activity of rat liver cytoplasm is low or absent in foetal animals but increases to adult levels 1–2 days after birth. In vitro experiments with isolated nuclei indicate that foetal nuclei have the capacity to accumulate dexamethasone but only when presented with cytosol-bound glucocorticoid.

The effect of glucagon on DNA synthesis in rat spleen and bone marrow

It has been demonstrated that glucagon will enhance the rate of radioactive thymidine incorporation into DNA in foetal and postnatal rat liver in vitro. The effectiveness of the hormone is age dependent, and declines postnatally [l] . There is a good correlation between the diminishing glucagon effect and the reduction in erythropoietic content of foetal rat liver [2,3] . During the development of the rat embryo, the sites of maximal erythropoietic activity are the yolk sac, the liver, the spleen and the bone marrow in chronological order [3] . This communication reports the results of experiments on the effect of glucagon on DNA synthesis in non-hepatic erythropoietic tissue in the rat.