Energy depletion and autophagy. Cytochemical and biochemical studies in isolated rat hepatocytes

Summary In this paper, data dealing with the sensitivity of autophagy towards partial ATP depletion in isolated rat hepatocytes are reviewed. Partial reduction of intracellular ATP causes: (1) a decrease of proteolytic flux; (2) a decrease in uptake of cytosolic components into the autophagic-lysosomal compartment; (3) either a decrease or no change in the ratio between volume densities of autophagosomes and lysosomes, depending on whether or not the cytosolic phosphate potential is affected; and (4) impairment of the lysosomal proton pump. It is concluded that the consecutive steps of autophagy all respond to relatively small changes of intracellular ATP concentration.     

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.     

Cadmium-induced hepatotoxicity in cultured rat hepatocytes as evaluated by morphometric analysis

Summary Freshly isolated hepatocytes from neonatal rats were cultured for approximately 24 h; incubated for 5, 30, or 60 min in solutions containing 0, 50, 100, or 200 μM cadmium; embedded in plastic; and sectioned for optical microscopy. The exeent of cadmium-induced hepatotoxicity was evaluated by double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of threedimensional information) whereby hepatocytes were classified on the basis of the severity of morphologic damage at the optical level. Both time and concentration effects were studied. Cultures exposed to 200 μM cadmium, for various intervals of time from 5 to 60 min, showed statistically significant reductions in the relative volume percent of normal hepatocytes, elevations (then reductions) in the relative volume percent of slightly damaged hepatocytes, increases in the relative volume percent of moderately damaged cells, and increases in the relative volume percent of severely damaged liver cells. As the concentration of cadmium was increased from 50 to 200 μM cadmium (during both 30 and 60-min exposures), significant trends were observed in cellular distribution patterns based on relative volume percent. Morphologically normal cells decreased, both slightly damaged and moderately damaged cells increased, and severely damaged cells remained unchanged. These results indicated that morphometric analysis at the optical level provided quantitative estimates for the evaluation of time- and concentration-effects of cadmium on cultured hepatocytes. This work was supported by a grant from the Johns Hopkins University Center for Alternatives to Animal Testing.     

Cadmium-induced hepatotoxicity in cultured rat hepatocytes as evaluated by morphometric analysis

Freshly isolated hepatocytes from neonatal rats were cultured for approximately 24 h; incubated for 5, 30, or 60 min in solutions containing 0, 50, 100, or 200 microM cadmium; embedded in plastic; and sectioned for optical microscopy. The extent of cadmium-induced hepatotoxicity was evaluated by double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of three-dimensional information) whereby hepatocytes were classified on the basis of the severity of morphologic damage at the optical level. Both time and concentration effects were studied. Cultures exposed to 200 microM cadmium, for various intervals of time from 5 to 60 min, showed statistically significant reductions in the relative volume percent of normal hepatocytes, elevations (then reductions) in the relative volume percent of slightly damaged hepatocytes, increases in the relative volume percent of moderately damaged cells, and increases in the relative volume percent of severely damaged liver cells. As the concentration of cadmium was increased from 50 to 200 microM cadmium (during both 30 and 60-min exposures), significant trends were observed in cellular distribution patterns based on relative volume percent. Morphologically normal cells decreased, both slightly damaged and moderately damaged cells increased, and severely damaged cells remained unchanged. These results indicated that morphometric analysis at the optical level provided quantitative estimates for the evaluation of time- and concentration-effects of cadmium on cultured hepatocytes.     

Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes

Seven cytosolic enzymes with varying half-lives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h; glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organelles (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeptin. Inhibitors of lysosomal fusion processes (vinblastine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolytic inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.     

Cytochemical localization of glutaraldehyde-resistant NAD(P)H-oxidase in rat hepatocytes

NAD(P)H-oxidase activity was demonstrated in glutaraldehyde-fixed rat hepatocytes by a cerium technique. The activity was observed exclusively on the bile-canalicular plasma membrane of hepatocyte. No reaction product was formed in the absence of NAD(P)H as the substrate. The reaction was inhibited by pCMB (surface sulfhydryl group specific reagent), by heating, by anaerobic incubation and by catalase (H2O2 scavenger), but it was not inhibited by KCN or NaN3. The present results show that bile-canalicular plasma membrane produces H2O2 and the cerium technique for demonstration of H2O2 is therefore an useful method for the subcellular localization of NAD(P)H-oxidase activity in the glutaraldehyde-fixed hepatocyte.     

Phospholipase C-evoked glycerol release in energy depleted rat myocardial cells

Summary Preincubation of rat myocardial cells in hypoxic substrate-free Krebs-Ringer bicarbonate buffer (pH 7.4, 37°C) resulted in a substantial decline in high energy phosphates (ATP and CP). Thus, 20 and 60 min preincubation produced a 18 and 72% decline in ATP content, whereas the parallel decline in CP content was 51 and 73%. This energy depletion was accompanied by a change in cell morphology from the initial rod-shaped form to rounded up (hyper-contracted) myocytes. In cells preincubated in substrate-free normoxic buffer, both normal morphology and energy homeostasis were maintained. When energy depleted myocytes later were incubated in the presence of phospholipase C (PLC), this resulted in a substantial release of glycerol, amounting to 92 and 137 nmol/10⁶ cells – 2 h in 20 and 60 min energy depleted myocytes, respectively. In addition, PLC caused an increased leakage of lactate dehydrogenase in energy depleted myocytes. Normal cells, on the other hand, were apparently not affected by PLC. These data suggest that PLC selectively attacks energy depleted and/or structurally damaged myocytes. This could well enhance the breakdown of the natural barrier between the extra- and intracellular compartments and thus augment the cellular damage during ischemia. Moreover, energy depleted myocytes appeared exceptionally sensitive to this enzyme, since the levels required to cause glycerol or lactate dehydrogenase release were several orders of magnitude lower than that required to cause membrane permeation in other cell types.     

Cytochemical determination of acid phosphatase activity in isolated rat hepatocytes during starvation-induced proteolysis

Summary Acid phosphatase activity in isolated rat liver parenchymal cells has been investigated with quantitative histochemical means during short-term starvation, which leads to a considerable loss in protein mass in the parenchyma. Animals trained to a meal-feeding regime in which food was available during 1 h only per 24 h (using an automatic food dispensing machine), were sacrified 6, 12, 18, 24 and 36 h after food had been withheld at the time point (23.00 h) of meal feeding. Acid phosphatase activity was analysed cytophotometrically in isolated hepatocytes incorporated into polyacrylamide gels before the enzyme reaction technique with the post-azo coupling was carried out. No indication could be found for any significant changes in the amount of acid phosphatase activity per individual hepatocyte during the entire period of fasting, as compared with two time points (11.00 and 23.00 h) before the theoretical onset of fasting. It is concluded that the considerable enhancement of protein degradation in the lysosomal apparatus during fasting is not reflected by changes in the cellular acid phosphatase activity.     

Adenylate energy charge of rat and human cultured hepatocytes

Summary A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908±0.008n=9, human: 0.918±0.014n=6, mean ± SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.     

Tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes

Mitochondria become targets for autophagic degradation after nutrient deprivation, a process also termed mitophagy. In this study, we used LysoTracker Red (LTR) and MitoTracker Green to characterize the kinetics of autophagosomal proliferation and mitophagy in cultured rat hepatocytes. Autophagy induced by nutrient deprivation plus glucagon increased LTR uptake assessed with a fluorescence plate reader and the number of LTR-labeled acidic organelles assessed with confocal microscopy in individual hepatocytes both by 4- to 6-fold. Serial imaging of hepatocytes coloaded with MitoTracker Green (MTG) revealed an average mitochondrial digestion time of 7.5 min after autophagic induction. In the presence of protease inhibitors, digestion time more than doubled, and the total number of LTR-labeled organelles increased about 40%, but the proportion of the LTR-labeled acidic organelles containing MTG fluorescence remained constant at about 75%. Autophagy inhibitors, 3-methyladenine, wortmannin and LY204002, suppressed the increase of LTR uptake after nutrient deprivation by up to 85%, confirming that increased LTR uptake reflected autophagy induction. Cyclosporin A and NIM811, specific inhibitors of the mitochondrial permeability transition (MPT), also decreased LTR uptake, whereas tacrolimus, an immunosuppressive reagent that does not inhibit the MPT, was without effect. In addition, the c-Jun N-terminal kinase (JNK) inhibitors, SCP25041 and SP600125, blocked LTR uptake by 47% and 61%, respectively, but ERK1, p38 and caspase inhibitors had no effect. The results show that mitochondria once selected for mitophagy are rapidly digested and support the concept that mitochondrial autophagy involves the MPT and signaling through PI3 kinase and possibly JNK.     

The relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes

The relationship between autophagy and the intracellular distribution of endocytosed asialoorosomucoid was studied in cultured rat hepatocytes. Overt autophagy was induced by shifting the cells to a minimal salt medium. Incubation in minimal salt medium led to the formation of buoyant lysosomes at the expense of denser lysosomes manifested as a dual distribution of these organelles in Nycodenz gradients. Asialoorosomucoid was labeled with 125I-tyramine cellobiose. The labeled degradation products formed from this ligand are trapped at the site of degradation and may therefore serve as markers for the subgroup of lysosomes involved in the degradation. In control cells the degradation of the ligand was initiated in a light prelysosomal compartment and continued in denser lysosomes. In cells with high autophagic activity, the degradation of labeled asialoorosomucoid took place exclusively in a buoyant group of lysosomes. These results suggest that degradation of endocytosed ligand takes place in the same secondary lysosomes as substrate sequestered by autophagic mechanisms. These light lysosomes represent a subgroup of active lysosomes which are gradually recruited from dense bodies. Data are also presented that indicate that insulin may prevent the change in buoyant density brought about by incubation in deficient medium.     

Cytochemical examination of the compartments involved in the transcellular transport of horseradish peroxidase in rat hepatocytes

Horseradish peroxidase (HRP, 10 mg/100 g body weight) was intravenously injected into rats in order to investigate the nature of the compartments involved in the transcellular transport of the protein through hepatocytes into bile. Double cytochemistry for HRP and the marker enzymes for cytoplasmic organelles was used. HRP was shown to be taken up by hepatocytes via vesicles at the sinusoidal surface, some of which were positive for 5'-nucleotidase activity. HRP was then found in the smooth-surfaced vesicles and tubules which were negative in 5'-nucleotidase, glucose 6-phosphatase, thiamine pyrophosphatase and acid phosphatase activity, suggesting that the tubular structures are neither the endoplasmic reticulum, the Golgi apparatus nor lysosomes. Biochemical studies revealed that the lead procedures used for the double cytochemistry did not inhibit the peroxidatic activity of HRP, and conversely that HRP did not interfere with the marker enzyme activity. Such cytochemical observations seemed to be supported by the observation that administration of monensin (3.5 mg/100 g) and chloroquine (5 mg/100 g), which markedly altered the structure of the Golgi apparatus and lysosomes, respectively, slightly altered the biliary excretion of HRP but not to a significant extent.     

Amino acid and energy requirements for rat hepatocytes in primary culture

The amino acid and energy requirements of rat hepatocytes in suspension and early culture were investigated. Among a number of potential energy substrates tested, pyruvate (20 mM) was found to be most effective in stimulating hepatocytic protein synthesis. Amino acids stimulated protein synthesis both as energy substrates and as protein precursors. An amino acid mixture was designed to provide maximal inhibition of protein degradation as well as maximal stimulation of protein synthesis. In a defined medium containing amino acids at these concentrations, and supplemented with glucocorticoid hormone and insulin, hepatocytes could be maintained--on a collagen substratum--for at least a week without any significant net loss of cells or cellular protein.     

Amino acid and energy requirements for rat hepatocytes in primary culture

Summary The amino acid and energy requirements of rat hepatocytes in suspension and early culture were investigated. Among a number of potential energy substrates tested, pyruvate (20 mM) was found to be most effective in stimulating hepatocytic protein synthesis. Amino acids stimulated protein synthesis both as energy substrates and as protein precursors. An amino acid mixture was designed to provide maximal inhibition of protein degradation as well as maximal stimulation of protein synthesis. In a defined medium containing amino acids at these concentrations, and supplemented with glucocorticoid hormone and insulin, hepatocytes could be maintained—on a collagen substratum—for at least a week without any significant net loss of cells or cellular protein. The work was supported by grants from The Norwegian Cancer Society and from The Norwegian Council for Science and the Humanities. An erratum to this article is available at .     

Hepatic autophagy and intracellular ATP. A morphometric study

In order to estimate the sensitivity of macroautophagy in liver toward changes in ATP we have analyzed the volume density of the autophagic/lysosomal system in isolated rat hepatocytes, incubated under conditions where intracellular ATP was partially depleted. (a) It appeared that reduction of the intracellular ATP concentration by 30-50% decreased the volume density of autophagic vacuoles by 70%. (b) Partial ATP depletion did not involve significant changes in the volume density of dense bodies. Together with studies showing that the rate of overall proteolysis via macroautophagy decreases with decreasing ATP concentration (P.J.A.M. Plomp, E.J. Wolvetang, A.K. Groen, A.J. Meijer, P.B. Gordon, and P.O. Seglen (1987) Eur. J. Biochem. 164, 197-203) our data indicate that changes in intracellular ATP primarily affect early steps in the autophagic/proteolytic pathway.     

Changes induced by fasting and cycloheximide in the vacuolar apparatus of rat hepatocytes. A morphometric investigation

An increase in the number of autophagic vacuoles and in the size of the dense and residual bodies was observed in the hepatocytes of rats fasted for 24 hours; moreover, the number of dense bodies was reduced. These data suggest that the previously reported acceleration in cell protein degradation caused by fasting can be accounted for by enhanced autography. The treatment with cycloheximide, which was previously found to prevent this proteolytic response, also prevents the appearance of signs of enhanced autophagic activity.