Newborn Rat Hepatocytes Research Articles

The administration of nonmetabolizable glucose analogues fails to suppress the development of glycogen autophagy in newborn rat hepatocytes

The effects of parenteral administration of glucose, 3-methylglucose (3MG), or 2-deoxyglucose (2DG) on the glycogen autophagy were studied in the newborn rat liver using electron microscopy and biochemical methods. The administration of glucose resulted in hyperglycemia and prevented the mobilization of hepatocytic glycogen. It also prevented the development of autophagic vacuoles in general and inhibited the glycogen-degrading activity of acid α-1,4-glucosidase. The nonphosphorylated and not further metabolized glucose analog 3MG also produced hyperglycemia, but increased acid glucosidase. Pretreating the newborns with the β-adrenergic blocker propranolol inhibited the effects of 3MG. The phosphorylated but not fully metabolized glucose analog 2DG produced similar effects. The administration of xylitol to the newborns already treated with 2DG, suppressed acid glucosidase. The results of this and our previous studies suggest that glucose must be metabolized beyond its phosphorylation step to inhibit acid glucosidase activity.

Autophagosomal glycogen‐degrading activity and its relationship to the general autophagic activity in newborn rat hepatocytes: The effects of parenteral glucose administration

The effects of the administration of parenteral glucose on the postnatal glycogen autophagic activity and its relationship to the general autophagic activity, were studied in newborn rat liver using electron microscopy and biochemical methods. Glucose abolished the normal postnatal hypoglycemia and preserved the hepatocytic hyaloplasmic glycogen to the levels of birth. It also inhibited the normal postnatal increase in the number and volume of autophagic vacuoles. Glucose especially decreased the rate of postnatal development of the glycogen-containing autophagic vacuoles. This decrease was greater than that of the autophagic vacuoles in general. In the control animals at the age of 6 h, the total volume of the glycogen-containing autophagic vacuoles accounted for 87% of the autophagic vacuoles in general, whereas in the glucose-treated animals of the same age, for only 62%. The results of this and previous studies support the view that the general autophagic activity that develops in the immediate postnatal period in rat hepatocytes is mainly expressed as glycogen autophagic activity selectively inhibited by glucose.

Intracrine signaling through lipid mediators and their cognate nuclear G-protein-coupled receptors: a paradigm based on PGE2, PAF, and LPA1receptorsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

Prostaglandins (PGs), platelet-activating factor (PAF), and lysophosphatidic acid (LPA) are ubiquitous lipid mediators that play important roles in inflammation, cardiovascular homeostasis, and immunity and are also known to modulate gene expression of specific pro-inflammatory genes. The mechanism of action of these lipids is thought to be primarily dependent on their specific plasma membrane receptors belonging to the superfamily of G-protein-coupled receptors (GPCR). Increasing evidence suggests the existence of a functional intracellular GPCR population. It has been proposed that immediate effects are mediated via cell surface receptors whereas long-term responses are dependent upon intracellular receptor effects. Indeed, receptors for PAF, LPA, and PGE2(specifically EP1, EP3, and EP4) localize at the cell nucleus of cerebral microvascular endothelial cells of newborn pigs, rat hepatocytes, and cells overexpressing each receptor. Stimulation of isolated nuclei with these lipids reveals biological functions including transcriptional regulation of major genes, namely c-fos, cylooxygenase-2, and endothelial as well as inducible nitric oxide synthase. In the present review, we shall focus on the nuclear localization and signaling of GPCRs recognizing PGE2, PAF, and LPA phospholipids as ligands. Mechanisms on how nuclear PGE2, PAF, and LPA receptors activate gene transcription and nuclear localization pathways are presented. Intracrine signaling for lipid mediators uncover novel pathways to elicit their effects; accordingly, intracellular GPCRs constitute a distinctive mode of action for gene regulation.

Intracellular Signaling of Lipid Mediators via Cognate Nuclear G Protein–Coupled Receptors

Platelet-activating factor (PAF) and lysophosphatidic acid (LPA) are ubiquitous lipid mediators that play important roles in inflammation, cardiovascular homeostasis, and immunity and are also known to modulate gene expression of specific proinflammatory genes. The mechanism of action of these phospholipids is thought to be primarily dependent on their specific plasma membrane receptors belonging to the superfamily of G protein-coupled receptors (GPCRs). However, increasing evidence suggests the existence of a functional intracellular GPCR population. It has been suggested that immediate effects are mediated by cell surface receptors, whereas long-term responses are mediated by intracellular receptors. PAF and LPA(1) receptors localize at the cell nucleus of cerebral microvascular endothelial cells of newborn pig, rat hepatocytes, and cells overexpressing each receptor, and stimulation of isolated nuclei reveal biological functions, including transcriptional regulation of major genes, namely cylooxygenase-2 and inducible nitric oxide synthase. This mini review focuses on the nuclear localization and signaling of GPCRs, recognizing PAF and LPA phospholipids as ligands. Theories on how nuclear PAF and LPA1 receptors activate gene transcription and nuclear localization pathways are discussed. Intracrine signaling for lipid mediators uncover novel pathways to elicit their effects; moreover, intracellular GPCRs constitute a distinctive mode of action for gene regulation.

Histochemical localization of acid mannose 6-phosphatase activity in the newborn rat hepatocytes

The localization of acid mannose 6-phosphatase activity in newborn rat hepatocytes was demonstrated at the electron microscopic level by using a histochemical method based on the work of Robinson and Karnovsky. Reaction product was virtually restricted to the lysosomes. Most of them exhibited various grades of reactivity. Some were devoid of activity. Our observations suggested that this histochemical method could be used to differentiate distinct subpopulations of lysosomes on the basis of their acid mannose 6-phosphatase activity.

Electron microscopic and biochemical study of the effects of rapamycin on glycogen autophagy in the newborn rat liver.

The effects of rapamycin on glycogen autophagy in the newborn rat liver were studied using biochemical determinations, electron microscopy, and morphometric analysis. Rapamycin increased the fractional volume of hepatocytic autophagic vacuoles, the liver lysosomal glycogen-hydrolyzing activity of acid glucosidase, the degradation of glycogen inside the autophagic vacuoles, and decreased the activity of acid mannose 6-phosphatase. These findings suggest that rapamycin, a known inhibitor of the mammalian target of rapamycin (mTOR) signaling, induces glycogen autophagy in the newborn rat hepatocytes. mTOR may participate in the regulation of this process.

An electron microscopic and biochemical study of the effects of propranolol on the glycogen autophagy in newborn rat hepatocytes.

The effects of propranolol on the glycogen autophagy in newborn rat hepatocytes were studied by using biochemical determinations, electron microscopy and morphometric analysis. Propranolol lowered the liver cyclic AMP and cyclic AMP-dependent protein kinase activity. It also decreased the formyl-methionyl-leucyl-phenylalanine (FMLP)-inhibitable Ca2+-ATPase activity including lysosomal calcium uptake pump. The normal postnatal increase in the volume of autophagic vacuoles and the activity of acid glycogen-hydrolyzing alpha glucosidase were inhibited. Also, the degradation of glycogen inside the autophagic vacuoles was apparently inhibited. The activity of acid mannose 6-phosphatase was increased. These findings indicate that propranolol influences several steps in the sequence of events leading to the breakdown of glycogen in the autophagic vacuoles of newborn rat hepatocytes. This supports our previous studies suggesting that cyclic AMP regulates glycogen autophagy.

Studies on glycogen autophagy: effects of phorbol myristate acetate, ionophore A23187, or phentolamine.

The effects of agents that could manipulate the lysosomal calcium such as phorbol myristate acetate, ionophore A23187, and phentolamine on the lysosomal glycogen degradation were studied by electron microscopy, morphometric analysis, and biochemical assays in newborn rat hepatocytes. Phorbol myristate acetate, which promotes the input of calcium to lysosomes, increased the total volume of autophagic vacuoles and the activity of lysosomal glycogen-hydrolyzing acid alpha 1,4 glucosidase and decreased the fractional volume of undigested glycogen inside the autophagic vacuoles and also decreased the activity of acid mannose 6-phosphatase. Ionophore A23187, which releases lysosomal calcium, produced opposite results in these enzyme activities. Phentolamine, an alpha-adrenergic blocking agent which interferes with the generation of phosphoinositides and may activate the lysosomal calcium uptake pump, increased the total volume of autophagic vacuoles and the activity of lysosomal glycogen-hydrolyzing acid glucosidase and decreased the fractional volume of undigested glycogen inside the autophagic vacuoles. The results of this study constitute evidence that changes in lysosomal calcium may influence certain aspects of autophagy, including the degradation of glycogen inside the autophagic vacuoles. They also support our previous postulate [Kalamidas and Kotoulas (2000a,b) Histol Histopathol 15:29-35, 1011-1018] that stimulation of autophagic mechanisms in newborn rat hepatocytes may be associated with acid mannose 6-phosphatase activity-deficient lysosomes.

Glycogen autophagy in newborn rat hepatocytes.

Glycogen autophagy in newborn rat hepatocytes was studied by using enzyme determinations and electron microscopy. Cyclic AMP induced glycogen autophagy in these cells. Glycogen-hydrolyzing acid glucosidase activity increased whereas acid mannose 6-phosphatase activity decreased in the liver of these animals. Parenteral glucose, which prevents postnatal glucagon secretion and tissue cyclic AMP elevation, and propranolol which antagonizes cyclic AMP, inhibited glycogen autophagy. Glucosidase activity decreased and phosphatase activity increased. These findings raise the possibility that cyclic AMP-induced autophagic mechanisms in newborn rat hepatocytes are associated with changes in the activity of acid mannose 6-phosphatase.

Studies on the breakdown of glycogen in the lysosomes: the effects of hydrocortisone.

The effects of hydrocortisone on newborn rat liver were studied by using biochemical assays, electron microscopy and quantitative morphometry. Hydrocortisone increased the number of lysosomes in the hepatocytes. Most of the lysosomes represented glycogen-containing autophagic vacuoles. The glucocorticoid also increased the activity of the liver glycogen-hydrolyzing acid glucosidase and the breakdown of glycogen inside lysosomes. The activity of the liver acid mannose 6-phosphatase was decreased. This may be related to the stimulation of autophagic mechanisms in the newborn rat hepatocytes.

Hepatocellular carcinoma cell lines from diethylnitrosamine phenobarbital-treated rats. Characterization and sensitivity to endothall, a protein serine/threonine phosphatase-2A inhibitor.

Primary hepatocellular carcinoma (HCC) is probably one of the most common fatal forms of liver cancer. We have established permanent cell lines from diethylnitrosamine/phenobarbital induced primary rat liver carcinomas to study new anticancer therapies. The rat hepatocellular carcinoma cell lines (HR-2, HR-3, and HR-4) have been maintained in culture for over 3 years. They form tumors when transplanted sc or im into young syngeneic rats. Immunocytology (alpha-fetoprotein, albumin), biochemical (gamma-glutamyl transferase), and histochemical (glycogen) marker studies and electron microscopy (biliary canaliculi) showed unique, stable differentiation patterns in these tumor lines. They overproduced the c-met protooncogene product and formed colonies spontaneously in semisolid culture with high cloning efficiency (HR-2: 50%-80%, HR-3: 35%-50% and HR-4: 50%-65%). The sensitivity of these cell lines to inhibitors of protein ser/thr phosphatase-2A (PP2A), a key enzyme in the control of G1/S and G2/M cell cycle phase transitions in eukaryotes, was studied in vitro. The specific, weak inhibitor of PP2A, endothall, caused dose- and time-dependent cytostasis specifically in G2/M. The cells died later by apoptosis, which was confirmed by cytology (annexin V-FITC labeling, propidium iodide painting of apoptotic bodies) and by fluorescent activated cell sorter (FACS) DNA measurements. The HR-2, HR-3, HR-4, and Zajdela hepatocellular carcinomas were most sensitive to endothall (IC50 of 1.7, 1.2, 0.9, and 1.7 microg/mL), whereas newborn rat hepatocytes growing exponentially in primary culture (IC50 = 6.2 microg/mL), rat DHD/K12 colon carcinoma cells (IC50 = 3.6 microg/mL), or human HT-29 colon carcinoma cells (IC50 = 4.9 microg/mL) were less sensitive. Thus, endothall inhibits preferentially HCC growth and these new rat hepatocellular carcinoma lines may be useful for further biochemical and pharmacological studies on PP2A inhibitors, and for testing new forms of treatment of hepatic cell carcinomas.

The degradation of glycogen in the lysosomes of newborn rat hepatocytes: glycogen-, maltose- and isomaltose-hydrolyzing acid alpha glucosidase activities in liver.

The lysosomal glucosidase activities and glycogen degradation in newborn rat liver were studied by using biochemical assays, electron microscopy and quantitative morphometry. Glycogen-hydrolyzing, maltose-hydrolyzing and isomaltose-hydrolyzing activities were low at birth but increased afterwards. At the age of 6 hours they were markedly elevated. Actinomycin prevented the development of glucosidase activities indicating that these depend on protein synthesis. Parenteral glucose inhibited all three activities. This was apparently due to the abolition of normal postnatal hypoglycemia and the need for blood glucose. Cyclic AMP increased the glycogen-hydrolyzing but not the maltose-hydrolyzing activity. Propranolol inhibited the glycogen-hydrolyzing but not the maltose-hydrolyzing activity. The observations of this study provide further support for the hypothesis made by previous investigators that these activities are due to different enzymes.

Long-term culture of adult rat hepatocyte spheroids

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of α 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.

Effect of conditioned medium of neonatal rat hepatocytes on mixed culture of kupffer cells and fibroblast-like liver cells

The study was carried out on primary coculture Kupffer cells and liver fibroblasts of newborn and mature rats. At first Kupffer cells and liver fibroblasts were taken on the equal quantity. There were observed significant decrease of Kupffer cells quantity and fibroblasts death after 4-5 days in coculture. Mitotic and functional activity liver fibroblasts, adhesion of Kupffer cells were increased by the using of conditioned medium of newborn rat hepatocytes. The rise of mitotic activity of liver fibroblasts and quantity of nucleolus in their nuclei forestalls of the increase Kupffer cells size, appearing of Kupffer cells with some nuclei.

Regulation on the Expression of Carcino-Embryonic Proteins in Newborn Rat Hepatocytes by Dexamethasone An Enzyme Histochemical and Immunohistochemical Study

Dexamethasone (DEX) was administrated intraperitoneally to newborn rats at a dose level of 2 micrograms/g body weight/day for four days, starting 4 days after birth. After administration of DEX, large quantities of glycogen granules accumulated in the cytoplasm of hepatocytes and the activity level of gamma-glutamyl transpeptidase (gamma-GTP) in liver homogenate increased significantly, whereas that of alkaline phosphatase (ALP) exhibited no significant difference in comparison with the control group. In the histochemical analysis, after administration of DEX, a high level of activity of gamma-GTP appeared along cell borders between adjacent hepatocytes in the peripheral portion of liver lobules. On the other hand, a low level of analysis, no hepatocyte showing positive reactions to Alpha-fetoprotein (AFP) could be recognized after administration of DEX, and in livers of newborn rats receiving DEX, the number of hepatocytes which incorporated bromodeoxyuridine (BUdR) decreased. The present study showed in newborn rat that DEX induced the differentiation of hepatocytes and regulated the expression of carcino-embryonic proteins.

Effect of L-Carnitine on Glycogen Synthesis and ATP Production in Cultured Hepatocytes of the Newborn Rat

Changes in intracellular carnitine, ATP and glycogen concentration were studied when hepatocytes of newborn or adult rats were incubated with oleate, lactate and/or carnitine. Hepatocytes of adult rats appeared to be able to maintain cellular carnitine concentration without exogenous carnitine supplementation. Hepatocytes of newborn rats appeared to be unable to maintain cellular carnitine concentration without carnitine supplementation. Moreover, ATP concentration and glycogen concentration were significantly increased by the carnitine supplement with oleate and/or lactate compared to the unsupplemented group. Increases in both intracellular ATP and carnitine concentration depended on the concentration of carnitine added to the medium. These results suggest that carnitine may be an important factor in glycogen synthesis and ATP production of newborn infants.

Effects of actinomycin D on the lysosomes of newborn rat hepatocytes.

The effects of actinomycin D on newborn rat liver were studied by using biochemical assays, electron microscopy, and quantitative morphometry. Actinomycin prevented the normal postnatal rise in acid a 1,4 glucosidase (maltase) activity and the breakdown of lysosomal glycogen. The results suggest that the postnasal increase in acid glucosidase activity is protein synthesis dependent. This enzyme is critical for the catabolism of the lysosomal glycogen in newborn rat hepatocytes.

Prenatal exposure to alcohol alters the Golgi apparatus of newborn rat hepatocytes: a cytochemical study.

The effect of prenatal exposure to ethanol on the Golgi apparatus of newborn rat hepatocytes has been studied cytochemically using several trans-Golgi markers (thiamine pyrophosphatase, uridine diphosphatase, inosine diphosphatase, acid phosphatase, and 5'-nucleotidase) as well as a cis-side marker (osmium impregnation). The amount of cerium phosphate formed in the cytochemical reactions was roughly quantitated by stereologic methods. The Golgi apparatus of about 40% of the hepatocytes appeared disorganized after alcohol treatment, and in the other 60%, the electron density of reaction product deposits for all phosphatases investigated was decreased. 5'-Nucleotidase was completely absent in cisternae of Golgi apparatus of treated cells. In control cells impregnated with osmium tetroxide, reduced osmium compounds were observed in most Golgi cisternae and in nearby vesicles. In contrast, only small vesicles appeared positive in treated hepatocytes. These results suggest that prenatal alcohol exposure alters some Golgi functions. Thus, the decrease in nucleoside diphosphatase and 5'-nucleotidase cytochemical activities after ethanol exposure strongly suggests that this treatment could affect glycosylation in the Golgi apparatus of newborn rat hepatocytes.

The effects of cyclic 3′,5′-AMP on the lysosomes of newborn rat hepatocytes

Certain effects of cyclic 3',5'-AMP administration on the lysosomes of newborn rat hepatocytes were studied using biochemical assays, electron microscopy, and quantitative morphometry. Cyclic AMP produced accelerations of postnatal hyaloplasmic glycogen breakdown and lysosomal glycogen breakdown, and an increase in activity of the lysosomal enzyme acid alpha-1,4-glucosidase (maltase). Ergotamine, a known antagonist of the effects of cyclic AMP, produced inhibitions of postnatal hyaloplasmic glycogen breakdown and lysosomal glycogen breakdown. Cyclic AMP increased while ergotamine decreased the volume of lysosomes. The results support the postulate that the catabolism of lysosomal glycogen is controlled by those agents that regulate the catabolism of hyaloplasmic glycogen (O. B. Kotoulas and M. J. Phillips, Amer. J. Pathol. (1971) 63, 1-17; O. B. Kotoulas et al., Amer. J. Pathol. (1971) 63, 23-36). Control is mediated by changes in the activity of the lysosomal enzyme acid alpha-1,4-glucosidase. Lysosomes actively participate in the degradation of hepatocellular glycogen.

CAMP-independent stimulation of glycogen phosphorylase in newborn rat hepatocytes.

Postnatal development of glycogen phosphorylase activation by the cAMP-independent pathway was examined in isolated rat hepatocytes from control and propylthiouracil-treated congenital hypothyroid rat pups. At 5 days postnatum there was complete phosphorylase activation by beta-adrenergic stimulation, glucagon, and the calcium ionophore A23187, but no activation by alpha-adrenergic stimulation. Activation of phosphorylase by angiotensin or vasopressin was less than in hepatocytes from adult rats (P less than 0.01). At 28 days postnatum activation by all of these hormones was complete. In the propylthiouracil-treated group hormone responsiveness was similar to the control at 5 days postnatum. However, alpha-adrenergic (P less than 0.025), angiotensin, and vasopressin (P less than 0.05) activation was decreased at 28 days postnatum, and beta-adrenergic, glucagon, and A23187 activation was complete. The attenuated responses were restored by thyroxine replacement from 15 days postnatum. [32P]Pi incorporation into phosphatidylinositol by epinephrine and vasopressin in 28-day propylthiouracil-treated rats was lower than the control (P less than 0.01). We speculate that the diminished phosphorylase response of hepatocytes to alpha-adrenergic, vasopressin, or angiotensin stimuli in the early neonatal period could be related to low receptor numbers and the weaker phosphoinositide response during this period. Also, the depressed phosphorylase response to alpha-adrenergic, vasopressin, and angiotensin stimulation in congenital hypothyroidism at 28 days postnatum could be related to a decrease in number of plasma membrane receptors for these agonists.