Hepatocytes Of Animals Research Articles

Vitamin A deficiency alters genomic expression for fibronectin in liver and hepatocytes.

Earlier work from our laboratory had shown that vitamin A-deficient rats had increased levels of fibronectin in their serum. To explain this result, we investigated the mechanism whereby vitamin A deficiency affects the production of fibronectin by liver and hepatocytes, since liver is the known source of plasma fibronectin. By use of cDNA specific for rat liver fibronectin, we showed that livers of vitamin A-deficient rats had a 2-4-fold increase in the level of fibronectin mRNA and also a higher transcription rate. Rate of synthesis and secretion of fibronectin was found to be increased 2-fold in primary cultures of hepatocytes of deficient animals. Exogenous addition of retinyl acetate or retinoic acid to the media reversed this increase to control levels. The increase was paralleled by an increase in fibronectin mRNA, also reversed by exogenous retinoic acid. At least 12 h were needed for this reversal to take place. Thus, vitamin A appears to regulate the synthesis of fibronectin through its action on fibronectin mRNA transcription. This represents the first reported observation of an action of vitamin A at the genomic level on the synthesis of a specific protein in liver.

Degeneration of granule cells following chronic phenytoin administration: An electron microscopic investigation of the mouse cerebellum

Fifteen male mice ( C57 Bl6J ) were fed the liquid diet “Stardit” supplemented with vitamins together with phenytoin for 8 weeks; experimental animals and controls were pair-fed. After 8 weeks of treatment, the anesthetized animals were perfused with 3.5% glutaraldehyde. Tissue samples of the cerebral cortex (area 3), cerebellum (vermis), thalamus, hypothalamus, and liver were embedded in Araldite. All phenytoin-treated animals displayed a hepatomegaly. Semithin sections and ultrastructural investigations of the cerebellar vermis showed pyknoses of granule cells and an enlargement and swelling of parallel fibers in presynaptic areas in the molecular layer. The swollen axons showed an accumulation of tubular structures which represented proliferated smooth endoplasmic reticulum. Similar tubular structures were observed in hepatocytes of experimental animals. It is proposed that phenytoin caused an induction of the microsomal system of hepatocytes and granule cells which led to a proliferation of the smooth endoplasmic reticulum. The transport of these organelles to the axon terminals of parallel fibers via the axoplasmic flow is assumed to cause a swelling of the presynaptic area. A dying-back process may then lead to pyknosis of granule cells. Chronic phenytoin administration to mice is a new experimental model of neuroaxonal dystrophy.

Increased Glucagon Receptors in Chronically Hypersomatotrophic and Hyperglucagonemic Rats

The effect of increased levels of growth hormone on glucagon binding by isolated hepatocytes and on the cellular cyclic AMP response to glucagon was evaluated in rats bearing growth hormone-secreting tumor (Mt-T-W15) and in rats treated with rat growth hormone. An increased binding, due to an increased number of receptors, was observed in both groups of animals. Glucagon binding did not correlate with plasma glucagon levels, suggesting a failure of down regulation, possibly due to an effect of growth hormone and insulin on the number of receptors. Tumor-bearing and growth hormone-treated rats had larger hepatocytes so that, when hormone binding was expressed in terms of square micrometer of membrane surface, it appeared decreased. When the tumor was removed the increase in the number of glucagon receptors per cell persisted, even though the average cell size returned toward normal. It is suggested that this retention of the receptors may have been the result of continuing hyperinsulinism. Basal cAMP levels were elevated in hepatocytes of tumor-bearing and growth hormone-treated animals, possibly due to cell hypertrophy. On the other hand, the maximum cAMP response to glucagon was not altered by the experimental procedures. A negative effect of insulin on cAMP accumulation may explain this apparent paradox. Indeed, hepatocytes isolated from rats following tumor removal, but with continuing hyperinsulinemia, had a lower maximum cAMP response, even though the glucagon binding per cell or per unit of cell surface was increased.

The contribution of hepatic metabolism to diet-induced thermogenesis

In order to examine the possible contribution of the liver to diet-induced thermogenesis, we examined the metabolism of hepatocytes from rats that had been fed a varied choice of highly palatable human food items (“cafeteria feeding”). Liver cells derived from cafeteria-fed rats that had been fasted for 20 hours showed marked increases in rates of respiration and gluconeogenesis in the presence of glycerol or sorbitol. These cells were also much less sensitive to the inhibitory effects of rotenone than were hepatocytes of control animals. Hepatocytes from fasted cafeteria-fed rats also demonstrated a substantially enhanced rate of fatty acid oxidation and ketogenesis, which did not appear to be correlated with cellular demands for adenosine triphosphate (ATP). This apparent fall in metabolic efficiency was confirmed by calorimetric studies, which indicated augmented cellular heat production. These changes in hepatic metabolism, associated with cafeteria-feeding, suggest that the liver may have a significant role in diet-induced thermogenesis.

Role of energy charge and redox state for hepatocyte gluconeogenesis of acutely uremic rats.

Hepatocytes were isolated from rats following bilateral nephrectomy, ureter ligation or sham operation under sodium pentobarbital (Nembutal) anesthesia to investigate the potential role of energy charge and redox state for the gluconeogenetic ability of liver cells. Ketogenesis from l-serine, sodium pyruvate or dihydroxyacetone was significantly higher in hepatocytes of acutely uremic rats indicating higher concentration of reducing equivalents in the mitochondria. During incubation, the mitochondrial redox state characterized by beta-hydroxybutyrate/acetoacetate ratio moved into direction of reduction in all experimental groups, whereas cytosolic redox state characterized by lactate/pyruvate ratio shifted to the oxidative state indicating lack of cytosolic reducing equivalents. Hepatocyte ATP and oxoglutarate production of ureter-ligated rats were significantly higher compared with binephrectomized or sham-operated animals independent of the substrates used. Simultaneously, energy charge showed values higher than 0.85 only in hepatocytes of ureter-ligated animals indicating high energy supply for energy requiring processes. We conclude that hepatic gluconeogenesis and ketogenesis of acutely uremic rats are limited by a lack of cytosolic reducing equivalents independent of cell energy supply.

Effect of fenofibrate and LF 2151 on hepatic peroxisomes in hamsters

Hamsters were given a diet containing fenofibrate (0.5% or 0.05%) or its metabolite, LF 2151 (0.15% or 0.015%) or a standard diet for a 3-week period. At the end of this period, the analysis of plasma lipids showed that the mean plasma triglyceride concentrations were not significantly different in the five groups of animals. The mean plasma cholesterol concentrations were significantly reduced in animals treated with both drugs but only when given at the high dosage. No consistent changes were noted in the liver weight/body weight ratio and the DNA content of the liver; the number of peroxisomes was increased in the hepatocytes of animals given fenofibrate at the high dosage. Liver homogenates were fractionated and the fractions rich in peroxisomes were used for assays of several enzymes involved in lipid metabolism. Compared with the control animals, activity of cyanide-insensitive fatty acyl-CoA (FA-CoA) oxidizing system was significantly increased by fenofibrate at the high dosage, carnitine acetyltransferase activity was markedly increased by both drugs at the high dosage and catalase activity remained unmodified. As there was a significant inverse correlation between the peroxisomal activity of FA-CoA oxidizing system and the plasma cholesterol concentrations, it is suggested that the increase of peroxisomal β-oxidation activity can be involved in the hypocholesterolemic action of fenofibrate and LF 2151. This is further substantiated by the finding that fenofibrate and LF 2151 were present in the peroxisomal fraction only in hamsters displaying hypocholesterolemia and high activity of FA-CoA oxidizing system. The presence of fenofibric acid in the plasma of hamsters given LF 2151 suggested that hepatocytes are able to generate the parent drug from this metabolite, underlining that the pharmacokinetics of fenofibrate are rather complex in hamsters.

Hydroxymethylglutaryl-coenzyme A reductase-containing hepatocytes are distributed periportally in normal and mevinolin-treated rat livers.

Mevinolin is a potent inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.34), an enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis. We have been studying the hepatic distribution of reductase with immunofluorescence microscopy and liver ultrastructure with electron microscopy in normal and drug-treated rats. In control animals, only about 20% of the hepatocytes were reductase positive. These cells were localized in the periportal lobular zones. The numbers of positive hepatocytes in animals given mevinolin or cholestyramine (or both) were directly proportional to the activities of the HMG-CoA reductase determined biochemically. This induction of HMG-CoA reductase immunofluorescence was centered periportally. Rats given 0.075% mevinolin alone had a homogeneous distribution of reductase staining in their hepatocyte cytoplasm, whereas a combination of 0.25% mevinolin and 3% cholestyramine caused a 150-fold increase in enzyme activity and induced prominent juxtanuclear immunofluorescent globules of HMG-CoA reductase in all hepatocytes. With electron microscopy, these bodies were composed of tightly packed stacks of smooth endoplasmic reticulum cysternae and aggregates of branched smooth endoplasmic reticulum tubules. Our data suggest that a subpopulation of periportal rat hepatocytes may be uniquely specialized for cholesterol synthesis.

Cytotoxicity of mononuclear cells and vulnerability of hepatocytes in alcoholic fatty liver of baboons.

Mononuclear cell cytotoxicity against autologous, allogeneic and xenogeneic (rabbit) hepatocytes was investigated in nine baboons fed alcohol for 17-21 months and in nine pair-fed controls. All alcohol-fed animals developed fatty liver. Cytotoxicity of mononuclear cells was not observed when rabbit hepatocytes were used as target cells, but mononuclear cells of alcohol-fed baboons were cytotoxic against hepatocytes of both control animals and hepatocytes from alcohol-fed baboons, including the animals' own hepatocytes. Increased vulnerability of hepatocytes of alcohol-fed baboons was also demonstrated since mononuclear cells of both controls and alcohol-fed animals were more cytotoxic against hepatocytes of alcohol-fed baboons than against those of controls. Thus, autologous and heterologous hepatocytes are more sensitive in the baboon than rabbit hepatocytes in demonstrating cytotoxicity already at the stage of fatty liver. Two factors are contributory: mononuclear cells cytotoxicity and vulnerability of hepatocytes.

Regulation of lipid synthesis in hepatocytes from lean and obese Zucker rats

Fatty acid synthesis and CO 2 production were evaluated in hepatocytes from lean and obese Zucker rats in the presence of 3H 2O, and several carbon precursors. The incorporation of 3H 2O into fatty acids was greater in obese compared to lean rats in both the isolated hepatocyte and in vivo. The rates of incorporation of 3H 2O into fatty acids and cholesterol in hepatocytes of both lean and obese rats were linear for 2 hr, in the absence or presence of 16.7 mM glucose. Rates of fatty acid synthesis were higher in the presence of 16.7 mM glucose compared to the absence of glucose in both lean and obese while rates of cholesterol synthesis were similar. The incorporation of 3H 2O into fatty acids, but not into cholesterol, was correlated with increasing glucose concentration and was 2 to three-fold higher in hepatocytes of obese compared to lean rats in the presence of several carbon precursors. Differences in CO 2 production between lean and obese rats suggested increased pentose phosphate shunt activity, decreased pyruvate dehydrogenase activity, and lower tricarboxylic acid cycle activity in obese rats. Fatty acid synthesis and CO 2 production from 3H 2O and [U- 14C]glucose in hepatocytes of lean and obese rats was similarly elevated by insulin and depressed by glucagon at several concentrations, suggesting that hepatocytes of obese animals respond to these hormones. These data indicate that rates of hepatic fatty acid synthesis although higher in obese rats respond to modulation in a fashion which is similar to the response in lean rats. The present studies suggest that the oxidation of several carbon precursors in the tricarboxylic acid cycle is diminished in obese compared to lean rats, but pentose phosphate shunt activity is greater in the obese Zucker rats.

Estrogen Regulation of Yolk and Non-Yolk Protein Synthesis in the Avian Liver: An Immunocytochemical Study

The effects of acute and chronic estrogen treatment on two egg yolk proteins, vitellogenin and apoVLDL-II, and two non-yolk proteins, ovalbumin and apoA-I, were studied by immunocytochemical techniques. Three groups of cockerels received either no treatment, or a single injection of diethylstilbestrol (DES, 2.5 mg) (acute stimulation) 24 h before killing, or 14 daily injections of 2.5 mg DES (chronic stimulation) before killing. The animals were killed at 4 weeks of age and their livers examined with respect to the distribution of the four different proteins by the indirect immunoperoxidase method. Vitellogenin was undetectable in the untreated cockerel liver. A single injection of DES resulted in the appearance of the protein in approximately 10%-15% of the hepatocytes. Chronic DES stimulation increased the number of positive cells to about 20%. In contrast, apoVLDL-II was present in 1%-2% of the hepatocytes in untreated animals. It was detected in an increased proportion (20%-25%) of cells after a single dose of DES. After chronic estrogen treatment, there was a very marked increase in the number of positive cells (less than 90%). Ovalbumin was undetectable in untreated cockerel liver, while apoA-I was detected in an extremely low proportion of cells (0.005%-0.01%). Neither ovalbumin nor apoA-I distribution seemed to be affected by a single dose of DES. However, chronic DES treatment resulted in the appearance of ovalbumin-containing cells (approximately 0.02%) and a marked increase in the number of cells containing apoA-I (10%-15%).

The stimulatory effect of alloxan-diabetes on the gluconeogenesis from alanine and glutamine in rabbit hepatocytes

1. 1. The stimulatory effect of alloxan-diabetes on the rate of gluconeogenesis from alanine and glutamine in rabbit hepatocytes was accompanied by both an increase in oxygen uptake and a decrease of the 3-hydroxybutyrate/acetoacetate ratio in hepatocyte suspension. 2. 2. Diabetes produced an acceleration of the transport of amino acids into hepatocytes as well as an elevation of their accumulation in the intracellular space. 3. 3. Activities of alanine aminotransferase, aspartate aminotransferase and glutaminase II were significantly increased in livers of diabetic animals, while those of both glutamate dehydrogenase and glutaminase I remained unaltered. 4. 4. Diabetes produced changes in the levels of gluconeogenic intermediates in hepatocytes incubated with alanine which were consistent with facilitation of reactions located in the gluconeogenic pathway between pyruvate and phosphoenolpyruvate and between fructose 1,6-bisphosphate and glucose. Increases in levels of first metabolites of gluconeogenic sequence were also observed in hepatocytes of diabetic animals incubated with glutamine. 5. 5. A decreased rate of lactate production from fructose in hepatocytes of diabetic rabbits was accompanied by a lower pyruvate kinase activity and an increased K 0.5 value of the enzyme for phosphoenolpyruvate in comparison with those determined in livers of fed animals. 6. 6. The data indicate that the stimulation of gluconeogenesis from alanine and glutamine in hepatocytes of alloxan-diabetic rabbits may result from: (i) an accelerated rate of transport of alanine and glutamine into cells; (ii) enhanced activities of aminotransferases: and (iii) a facilitation of reactions of first steps of gluconeogenic sequence and of those catalyzing the conversion of fructose 1,6-bisphosphate to glucose.

Morphology of nuclear ribonucleoproteins in the early period of liver regeneration after irradiation.

Cytochemical study of hepatocytes nuclei of the liver irradiated with a dose of 650 rad and irradiated after partial hepatectomy was performed. Morphological exponents of the intensive synthesis of preribosomal RNA in the nucleolus, as well as the transcription of heterogeneous RNA contained in perichromatin fibrils were revealed under the ionizing radiation effect. The behavior of perichromatin granules in the course of the experiment suggests delay in the transport of messenger RNA to cytoplasm, influenced by gamma radiation. Nuclear bodies were observed very often in nuclei of hepatocytes of irradiated animals.

Cellular basis of induced alpha-fetoprotein synthesis by hepatocytes of adult mouse after hepatotoxic injury and partial hepatectomy.

AbstractThe dynamics of α‐fetoprotein (AFP) production by hepatocytes following different experimental manoeuvres that are known to induce regeneration of the liver was studied in young adult mice by simultaneous identification of dividing cells arrested during mitosis and of cells containing immuno‐histochemically detectable AFP. After 70% hepatectomy AFP was produced by a few, largely periportally located hepatocytes, following waves of brisk and active cell division. Carbon tetrachloride (CCl4)‐induced hepatic injury, on the other hand, led to participation of a relatively larger number of cells in early AFP synthesis preceding a less extensive cell proliferation. Initially, AFP‐producing cells were located in the peripheral part of the lobule but later they were more centrally placed, most often surrounding the zone of necrosis. Cells in mitosis often contained large amounts of AFP. CCl4 administration 48 h after 70% hepatectomy resulted in extensive AFP production by approximately a quarter of all surviving liver cells, although the concurrent rate of cell division was low. Intoxication with dimethylnitrosamine showed a pattern identical to that of hepatectomy but both cell division and AFP production were of comparatively lower magnitude. It is concluded that at least two distinct modes exist for augmented synthesis of AFP by hepatocytes of adult animals. In one, pre‐existent cells are stimulated to produce the protein before and irrespective of cell division while in the other, small amounts are synthesized by newly generated cells. Combination of the two mechanisms results in high levels of AFP production by stimulation of a large number of young hepatocytes.

Sublethal effects of a pure polychlorobiphenyl on mice

Sexually mature, weight-stable male mice were dosed by oral intubation with either 0, 200, 500, or 1000 mgm/kgm of 2,4,5,2′,4′,5′-hexachlorobiphenyl (PCB), dissolved in peanut oil, and housed for 28 days in groups of five in metabolic cages. Their food and water consumption, urine and fecal (dry) output, and body weights were not depressed or enhanced by the PCB treatment; nor were any neuromuscular deficiencies detected by rotarod tests. At the termination of the experiment, hematocrit values were not significantly changed. The wet weights (percentage of body weight) of whole brain, testes, and epididymal fat pads were not changed significantly in the PCB-treated animals. However, wet spleen weights of those animals receiving 500 mgm of PCB/kgm were significantly depressed and the wet kidney weights of animals receiving 500 and 1000 mgm of PCB/kgm were also decreased significantly. The wet and dry weights of livers were significantly elevated in animals treated with 500 and 1000 mgm of PCB/kgm. The average amount of PCB extracted from dry livers increased significantly with each higher dose of PCB administered. Linear regression analysis revealed a dose-related response in the amount of PCB sequestered in the liver. The polychlorinated biphenyl extracted from dry livers was confirmed, by mass spectral analysis, to be 2,4,5,2′,4′,5′-hexachlorobiphenyl. Concerning cytologic changes in hepatocytes, the mean cell volume, mean nuclear volume, and mean cytoplasmic volume of centrilobular hepatocytes treated with 200 mgm of PCB/kgm (and higher) were all significantly greater than these same parameters in the centrilobular hepatocytes of untreated animals. No cytologic volume changes were detected in the periportal hepatocytes. Morphometric analysis of electron micrographs (35,000x) from each of 11 mice (three mice given 200 mgm of PCB/kgm and four mice each given 0 or 1000 mgm of PCB/kgm) revealed that the number of lipid bodies and mitochondria per cell were significantly higher in the average centrilobular hepatocyte treated with 1000 mgm of PCB/kgm. The membrane surface areas (expressed as square microns) were also significantly elevated in the mitochondrial envelope, the mitochondrial cristae, and the smooth endoplasmic reticulum per centrilobular hepatocyte. The volumes (expressed as cubic microns) of numerous cytoplasmic components were significantly greater in the centrilobular hepatocytes treated with 1000 mgm of PCB/kgm; these include lipid bodies, secondary lysosomes, mitochondria, rough and smooth endoplasmic reticulum, and open cytoplasm. In conclusion, this study gives a detailed quantitative morphometric description of the way in which centrilobular hepatocytes adapt to the toxic effects of a persistent environmental contaminate, 2,4,5,2′,4′,5′-hexachlorobiphenyl.

Changes in mitotic activity of hepatocytes and resorption of necrotic areas during induction of cirrhosis of the liver

Cirrhosis of the liver was induced in rats weighing 120–140 g by administration of CCl4 for 2 months. Iodinated oil was injected through the spleen of the experimental and intact animals to produce embolism of the branches of the portal vein and foci of necrosis in the liver tissue. The volume of the necrotic foci and the mitotic activity of the hepatocytes were determined. In cirrhosis of the liver the necrotic foci were resorbed more rapidly. The increase in the mitotic index on the second day after injection of the iodinated oil was greater in the control rats. The results suggest the appearance of a new cell clone in the liver which is responsible for resorption of the necrotic tissue and the reduction in mitotic activity of the hepatocytes in animals during development of cirrhosis of the liver.

Ultrastructural studies of the hepatocytes after chronic exposure to low levels of halothane

Adult rats were exposed to 10 ppm or 500 ppm halothane 8 hr/day and 5 days/wk for 8 wk or 4 wk, respectively. In the liver from animals which were exposed to 10 ppm of halothane, the rough endoplasmic reticulum in some hepatocytes accumulated a floccular, electron-dense material which gave the hepatocytes a dense and dark appearance. Increase in the matrical density and C-shaped transformation were observed in the mitochondria of some hepatocytes. In addition to these findings, areas of focal cytoplasmic degradation, dilatation of the bile canaliculi, peribiliary accumulation of lysosomes, and extensive dilatation of the smooth endoplasmic reticulum to form large cytoplasmic vacuoles were also observed in the hepatocytes of animals which had been exposed to 500 ppm halothane. Toxic potential of halothane upon chronic exposure is suggested.

Effect of pregnenolone-16α-carbonitrile, a microsomal enzyme inducer, on the regenerating rat liver

PCN, a microsomal enzyme inducer, given orally (10 mg in 1 ml water twice daily for 5 days), increased liver weight and mitotic activity in intact as well as in partially hepatectomized rats. Electron microscopy revealed SER proliferation in the hepatocytes of animals treated with PCN alone. Accumulation of SER membranes was also evident in the liver cell cytoplasm of untreated, partially hepatectomized rats; it was however, more pronounced in the hepatocytes of partially hepatectomized animals given PCN. These results indicate that the steriod has a marked effect on the regeneration rat liver.