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.     

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.     

Glycogen autophagy in the liver and heart of newborn rats. The effects of glucagon, adrenalin or rapamycin

The effects of glucagon, adrenalin or rapamycin on glycogen autophagy in the liver and heart of newborn rats were studied using biochemical determinations and electron microscopy. Glucagon or adrenalin increased autophagic activity in the hepatocytes and myocardiocytes, glycogen-hydrolyzing acid glucosidase activity in the liver and heart and degradation of glycogen inside the autophagic vacuoles. Glucagon or adrenalin also increased the maltose-hydrolyzing acid glucosidase activity in the liver, but not in the heart. Similar effects were produced in the newborn heart by rapamycin. These observations support previous studies suggesting that the cellular machinery which controls glycogen autophagy in the liver and heart of newborn animals, is regulated by the cyclic AMP and the mTOR pathways.     

Modifications of liver lysosomes in diabetic rats.

Recently investigators reported ultrastructural modifications of rat liver lysosomes which probably correlate with an increased level of blood plasma glucagon in streptozotocin-diabetes. We are investigating whether biochemical changes occur in this condition. Lysosome fragility is increased in the hepatocytes of streptozotocin-diabetic rats. Moreover the plasma activity of two glycosidases, B-N-acetylglucosaminidase and B-galactosidase, is markedly increased in streptozotocin-treated rats. Both these changes are largely prevented by insulin treatment. These findings support the idea that the morphologic and biochemical modifications of the hepatocytes, which are observed in experimental diabetes, involve the lysosomes showing an increased autophagic activity which is probably connected with enhanced liver protein catabolism.     

Translocation of hepatocyte lysosomes following partial hepatectomy and its inhibition by colchicine

Hepatocyte microtubules were studied during the translocation of lysosomes which follows partial hepatectomy. In hepatocytes of normal rat liver the lysosomes are seen mainly along the bile canaliculi. After partial hepatectomy, these lysosomes lose their pericanalicular arrangement and move towards large inclusions (‘protein droplets’) which result from the marked pinocytosis induced by the removal of about two-thirds of the liver mass. The lysosomes fuse with the inclusions, and by 4 h after partial hepatectomy most of the inclusions demonstrate acid phosphatase activity histochemically. Many microtubules are found in close proximity to the lysosomes. When the rats are treated with colchicine prior to the partial hepatectomy, events are markedly different. The lysosomes change their location but do not hit the cytoplasmic inclusions, and the inclusions remain negative for acid phosphatase activity even 4 h post-hepatectomy. Quantitative ultrastructural study shows that the volume density of microtubules in the hepatocytes decreases to one-third of that in control hepatocytes. This strongly suggests an involvement of microtubules in giving orientation to the translocation of hepatocyte lysosomes under these conditions.     

Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver

Summary Morphologically detectable protein (intramembrane particles) and cholesterol (filipin labelling) in the membranes of autophagic vacuoles and lysosomes were studied in mouse hepatocytes using thin-section and freeze-fracture electron microscopy. Both isolated autophagic vacuoles and lysosomes, and intact tissue blocks were used due to the facts (i) that lysosomes are difficult to recognize in freeze-fracture replicas of intact hepatocytes, and (i) that filipin penetration into the tissue blocks is unsatisfactory. Intramembrane particle density was low in the membranes of early autophagic vacuoles (defined as round-shaped vacuoles in which an inner membrane parallel with the outer limiting membrane was clearly visible). The lysosomal membranes contained considerably more intramembrane particles. Particle-rich lysosomes or other vesicles were observed to fuse with the early autophagic vacuoles. The membranes of nascent autophagic vacuoles with morphologically intact contents were usually not labelled by filipin, whereas the membranes of all other autophagic vacuoles and lysosomes were heavily labelled. The increased cholesterol in the membranes of slightly older autophagic vacuoles is presumably derived from cholesterol-rich lysosomes or other vesicles fusing with the vacuoles and from the degrading organelles inside the autophagic vacuoles.     

Cytochemical and morphometric analysis of autophagy in energy depleted rat hepatocytes

The energy dependence of lysosomal enzyme acquisition by autophagosomes was studied in isolated rat hepatocytes by ultrastructural analysis for acid phosphatase activity. Reduction of the intracellular ATP content by addition of atractyloside or fructose decreased the flux through the autophagic proteolytic pathway to a similar extent (40-50%). Unexpectedly, in the presence of atractyloside the volume density of autophagosomes was reduced by 65%, whereas in the presence of fructose this reduction was only 20%. The volume density of lysosomes was not significantly affected by either of the two compounds. It is concluded that partial ATP depletion by fructose not only inhibits sequestration of cytoplasmic material in autophagosomes, but also affects the fusion between autophagosomes and lysosomes. Since fructose, in contrast to atractyloside, does not affect the cytosolic phosphate potential, it is proposed that autophagic sequestration is more sensitive to changes in the cytosolic phosphate potential whereas the fusion between autophagosomes and lysosomes is more responsive to changes in the ATP concentration.     

Isolation and characterization of autophagic vacuoles from rat kidney cortex

The number of autophagic vacuoles in the proximal tubule cells of the rat kidney increased considerably after 3 h of vinblastine treatment. This increase was paralleled by stimulated proteolysis in an homogenate prepared from the cortex. We have taken advantage of this expansion in autophagic vacuoles in an effort to isolate these organelles from rat kidney cortex on a discontinuous Metrizamide gradient. Autophagic vacuoles have recently been purified from liver but not from other tissues. The purity of the isolated fraction was 95% of which 55% consisted of typical intact autophagic vacuoles containing sequestered organelles and 45% of other types of secondary lysosome. On plane section many of these displayed one or several intramatrical vesicles or flap like processes forming apparent vesicles at the pole of the organelles, which occasionally contained pinocytosed membranous material. These lysosomes were designated microautophagic vacuoles. It is suggested that the microautophagic vacuoles could be the morphological expression of uptake into lysosomes of small portions of cytosol. The isolated autophagic vacuole fraction was enriched in lysosomal enzymes (acid phosphatase and cathepsin D activities) and displayed high proteolytic rates, especially at acid pH.     

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.     

Rates of RNA degradation in isolated rat hepatocytes. Effects of amino acids and inhibitors of lysosomal function.

1. RNA degradation in isolated rat hepatocytes was measured as the release of radioactive cytidine from fed rats previously labeled in vivo for 60 h with [6-14C]orotic acid. Rates were determined from the linear accumulation of [14C]cytidine between 30 and 120 min of incubation in the presence of 0.5 mM unlabeled cytidine to suppress reutilization. 2. In the absence of amino acids, rates of RNA degradation in isolated hepatocytes averaged 3.97%/h. A complete mixture of amino acids added at 10-20 times normal plasma concentration inhibited RNA degradation by 65-70%. However, at physiological concentrations of amino acids, RNA degradation in isolated rat hepatocytes was less responsive as compared to perfused rat livers. 3. Numerous and large autophagic vacuoles at various stages of digestion were identified throughout the cytoplasm of isolated hepatocytes after 2 h of incubation in the absence of amino acids. The addition of amino acids at 20 times normal plasma concentration abolished almost completely the appearance of autophagic vacuoles. Furthermore, prophylamine, which accumulates in lysosomes, suppressed RNA degradation by 65% and the inhibitor of autophagic vacuole formation, 3-methyladenine, inhibited 70-80% of the degradation. Taken together, these results strongly suggest a contribution of the lysosomal system in the increase of RNA degradation rates in isolated rat hepatocytes.     

Use of digitonin extraction to distinguish between autophagic-lysosomal sequestration and mitochondrial uptake of [14C]sucrose in hepatocytes.

In isolated rat hepatocytes, electroinjected [14C]sucrose is sequestered both by mitochondria and by autophagosomes/lysosomes. Radioactivity can be selectively extracted from the latter organelles by low concentrations of digitonin, thereby providing a specific bioassay for autophagic sequestration. By including a digitonin extraction step in the assay procedure, autophagic [14C]sucrose sequestration could be shown to be virtually completely (greater than 90%) suppressed by the autophagy inhibitor 3-methyladenine (10 mM), whereas mitochondrial sugar uptake was unaffected. An amino acid mixture likewise suppressed autophagic sequestration very strongly, while having no detectable effect on the mitochondria.     

Quantitative analysis of immunogold labelling for ferritin in liver from control and iron-overloaded rats

Summary The distribution of ferritin antigenicity in control and iron-loaded rat hepatocytes was investigated with an immunogold-ferritin antibody technique. Antibody to horse spleen ferritin showed immunoreactivity as determined by dot blotting with immunogold/silver staining with purified rat liver ferritin but not with rat haemosiderin. The initial site of ferritin degradation was studied by analysing the density of gold labelling in the cytosol and lysosomes in combination with pre-embedding acid phosphatase cytochemistry.Immunoreactive ferritin was present in the cytosol, cytosolic clusters and lysosomes of normal hepatocytes. After iron-loading, the labelling density increased over tenfold in parenchymal cell cytosol with a smaller increase in Kupffer cells. Ferritin clusters contained substantially more immunoreactive ferritin than equivalent areas of lysosomes or cytosol. Analysis of the labelling density in hepatocyte lysosomes showed that, despite a striking increase in iron content, one-quarter of the lysosomes showed less immunolabelled ferritin than the cytosol. The existence of a wide range of ferritin labelling densities in the lysosomes with a large proportion unlabelled suggests that the ferritin protein shell is not degraded at a significant rate either in the cytosol or in clusters but only after incorporation into lysosomes.     

Morphometric analysis of the autophagic and crinophagic lysosomal systems in mammotropes throughout the estrous cycle of the rat

Summary The crinophagic and autophagic lysosomal systems were studied in mammotropes (prolactin secreting cells) of the adenohypophysis throughout the estrous cycle of the rat. By means of morphometric analysis, it was found that the volume of secondary autophagic lysosomes was usually greater than that of the crinophagic type. Although the volumes of both secondary autophagic and crinophagic lysosomes were minimal throughout proestrus and diestrus 2, the autophagic lysosomal volume per mammotrope was elevated during the estrous period. The volume of secondary crinophagic lysosomes per mammotrope increased during late estrus and remained elevated throughout early diestrus 1. Furthermore, there was an inverse relationship between the volume of mature secretory granules per cell and of the crinophagic system. These data suggest a role for lysosomes in the regulation of synthesis and secretion of prolactin by the adenohypophysis of the rat.Supported by grant HD 11571 from the National Institutes of Health     

Concanavalin A/IFN-gamma triggers autophagy-related necrotic hepatocyte death through IRGM1-mediated lysosomal membrane disruption

Interferon-gamma (IFN-γ), a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A) can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP) is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1) translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/-) mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.     

Influence of insulin on lysosomal activity and urea production in isolated parenchymal cells from rat liver.

Non-latent (free) activities of two lysosomal enzymes (acid phosphatase and beta-glucuronidase) and urea production were measured in purified rat liver parenchymal cells incubated in the presence and absence of insulin. Non-latent enzyme activity was measured by including 0.25M sucrose in the assay mixtures to provide osmotic protection to the lysosomes. Total enzyme activity was estimated with Triton X-100 in the homogenates. Insulin was found to inhibit ureogenesis and to reduce non-latent lysosomal enzyme activity in the hepatocytes in vitro. Our data support the idea that insulin inhibits autophagy in rat liver parenchymal cells. Such an effect of insulin may also explain the inhibitory action of insulin on urea production in the rat liver.     

Role of LAMP-2 in lysosome biogenesis and autophagy

In LAMP-2-deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2-deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2-deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation.     

Immunocytochemical study of lysosomal proteins with a new monoclonal antibody directed against epithelioid macrophages

 A monoclonal antibody, EPI-1, was produced against macrophages in epithelioid granulomas induced in rat foot pads by muramyl dipeptide. This EPI-1 antibody reacted to lysosome-like structures in epithelioid macrophages, peritoneal and pulmonary macrophages, and also in other tissues such as liver, testes, and kidneys. Western blot analysis of epithelioid granulomas, liver, testes, and kidneys revealed the same positive band of 62 kDa. Immunoelectron microscopic study of foot pad granulomas and hepatocytes demonstrated the EPI-1 antigen located in lysosomes and autophagic vesicles, preferentially along their membranes. These findings suggest that the EPI-1 antibody may recognize a novel antigen related to lysosomal membrane proteins in macrophages and other cells, which is useful for identifying lysosomes and their related structures. Accepted: 14 July 1997     

The effect of a conditioned medium from neonatal rat hepatocytes on a 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.     

Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase,legumain

To investigate the stepwise autophagic-lysosomal processing of hepatocellular proteins, the abundant cytosolic enzyme, betaine:homocysteine methyltransferase (BHMT) was used as a probe. Full-length (45 kDa) endogenous BHMT was found to be cleaved in an autophagy-dependent (3-methyladenine-sensitive) manner in isolated rat hepatocytes to generate a novel N-terminal 10-kDa fragment (p10) identified and characterized by mass spectrometry. The cleavage site was consistent with cleavage by the asparaginyl proteinase, legumain and indeed a specific inhibitor of this enzyme (AJN-230) was able to completely suppress p10 formation in intact cells, causing instead accumulation of a 42-kDa intermediate. To prevent further degradation of p10 or p42 by the cysteine proteinases present in autophagic vacuoles, the proteinase inhibitor leupeptin had to be present. Asparagine, an inhibitor of amphisome-lysosome fusion, did not detectably impede either p42 or p10 formation, indicating that BHMT processing primarily takes place in amphisomes rather than in lysosomes. Lactate dehydrogenase (LDH) was similarly degraded primarily in amphisomes by leupeptin-sensitive proteolysis, but some additional leupeptin-resistant LDH degradation in lysosomes was also indicated. The autophagic sequestration of BHMT appeared to be nonselective, as the accumulation of p10 (in the presence of leupeptin) or of its precursors (in the additional presence of AJN-230) proceeded at approximately the same rate as the model autophagic cargo, LDH. The complete lack of a cytosolic background makes p10 suitable for use in a “fragment assay” of autophagic activity in whole cells.

Incubation of hepatocytes with ammonium chloride, which neutralizes amphisomes as well as lysosomes, caused rapid, irreversible inhibition of legumain activity and stopped all p10 formation. The availability of several methods for selective targeting of legumain in intact cells may facilitate functional studies of this enigmatic enzyme, and perhaps suggest novel ways to reduce its contribution to cancer cell metastasis or autoimmune disease.     

Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase, legumain

To investigate the stepwise autophagic-lysosomal processing of hepatocellular proteins, the abundant cytosolic enzyme, betaine:homocysteine methyltransferase (BHMT) was used as a probe. Full-length (45 kDa) endogenous BHMT was found to be cleaved in an autophagy-dependent (3-methyladenine-sensitive) manner in isolated rat hepatocytes to generate a novel N-terminal 10-kDa fragment (p10) identified and characterized by mass spectrometry. The cleavage site was consistent with cleavage by the asparaginyl proteinase, legumain and indeed a specific inhibitor of this enzyme (AJN-230) was able to completely suppress p10 formation in intact cells, causing instead accumulation of a 42-kDa intermediate. To prevent further degradation of p10 or p42 by the cysteine proteinases present in autophagic vacuoles, the proteinase inhibitor leupeptin had to be present. Asparagine, an inhibitor of amphisome-lysosome fusion, did not detectably impede either p42 or p10 formation, indicating that BHMT processing primarily takes place in amphisomes rather than in lysosomes. Lactate dehydrogenase (LDH) was similarly degraded primarily in amphisomes by leupeptin-sensitive proteolysis, but some additional leupeptin-resistant LDH degradation in lysosomes was also indicated. The autophagic sequestration of BHMT appeared to be nonselective, as the accumulation of p10 (in the presence of leupeptin) or of its precursors (in the additional presence of AJN-230) proceeded at approximately the same rate as the model autophagic cargo, LDH. The complete lack of a cytosolic background makes p10 suitable for use in a "fragment assay" of autophagic activity in whole cells. Incubation of hepatocytes with ammonium chloride, which neutralizes amphisomes as well as lysosomes, caused rapid, irreversible inhibition of legumain activity and stopped all p10 formation. The availability of several methods for selective targeting of legumain in intact cells may facilitate functional studies of this enigmatic enzyme, and perhaps suggest novel ways to reduce its contribution to cancer cell metastasis or autoimmune disease.