Glycogen autophagy in glucose homeostasis.

Glycogen autophagy, the sequestration and degradation of cell glycogen in the autophagic vacuoles, is a selective, hormonally controlled and highly regulated process, representing a mechanism of glucose homeostasis under conditions of demand for the production of this sugar. In the newborn animals, this process is induced by glucagon secreted during the postnatal hypoglycemia and inhibited by insulin and parenteral glucose, which abolishes glucagon secretion. Hormonal action is mediated by the cAMP/protein kinase A (induction) and phosphoinositides/mTOR (inhibition) pathways that converge on common targets, such as the protein phosphatase 2A to regulate autophgosomal glycogen-hydrolyzing acid glucosidase and glycogen autophagy. Intralysosomal phosphate exchange reactions, which are affected by changes in the calcium levels and acid mannose 6- and acid glucose 6-phosphatase activities, can modify the intralysosomal composition in phosphorylated and nonphosphorylated glucose and promote the exit of free glucose through the lysosomal membrane. Glycogen autophagy-derived nonphosphorylated glucose assists the hyaloplasmic glycogen degradation-derived glucose 6-phosphate to combat postnatal hypoglycemia and participates in other metabolic pathways to secure the fine tuning of glucose homeostasis during the neonatal period.

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.

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 glucagon on glycogen autophagy in the liver and heart of newborn rats.

The effects of glucagon on the ultrastructural appearance and acid glucosidase activities in the liver and heart of newborn rats were studied. Liver or heart glycogen-hydrolyzing activity of acid glucosidase increased 3 hours after birth and gradually decreased from 3 to 9 hours. Maltose-hydrolyzing activity of acid glucosidase also rose 3 hours after birth, maintained a plateau between 3 and 6 hours, and fell at 9 hours. The administration of glucagon increased autophagic activity in the hepatocytes at the age of 6 hours. Glycogen inside the autophagic vacuoles was decreased, apparently due to the increased glycogen degradation. Glycogen-hydrolyzing activity was elevated in both the liver and the heart. Maltose-hydrolyzing activity was elevated in the liver, but not in the heart. The results of this study suggest that the glycogen-hydrolyzing and maltose-hydrolyzing activities of acid glucosidase are due to different enzymes. Glucagon's effect on the glycogen-hydrolyzing acid glucosidase activity and autophagosomal morphology is similar in both the liver and the heart.

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.

In vitro effects of hormones and autacoids on the activity of acid phosphatase in the lysates of endotoxin-activated rat peritoneal and bronchoalveolar macrophages.

Peritoneal and bronchoalveolar macrophages activated in vitro by endotoxin, exhibit alterations in the acid phosphatase activity of cell lysates when certain hormones or autacoids are present in the culture medium. They also show morphological changes concerning general appearance and acid phosphatase cytochemistry. Certain agents known to increase the intracellular levels of cyclic AMP, such as dopamine and prostaglandin E2, decreased this enzyme activity in the lysates of peritoneal macrophages. Adrenalin had no effect on this activity at 14 hours, but was found to increase the activity in the culture medium at the initial hours of incubation. Glucagon decreased whereas insulin increased acid phosphatase activity in bronchoalveolar macrophages. Serotonin or histamine, known to activate phospholipase C, increased this activity in peritoneal or bronchoalveolar macrophages. The results of this study, taken together with previously published data (Kondomerkos et al., 2003), suggest that hormones and autacoids may control certain parameters of macrophage activation including acid phosphatase activity.

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.

In vitro effects of hormones and autacoids on the hydrogen peroxide production and the morphology of endotoxin-activated rat peritoneal macrophages.

Peritoneal macrophages activated in vitro by endotoxin exhibit alterations of their capability to produce hydrogen peroxide after phorbol ester stimulation when certain hormones or autacoids are present in the culture medium. They also show morphological changes, mainly concerning cell size and nuclear appearance. Agents known to increase the intracellular levels of cyclic AMP, e.g. adrenalin and PGE2 reduce the hydrogen peroxide production. Insulin, which is known to decrease cyclic AMP levels, produces opposite results. Agents postulated to act via phospholipase C, e.g. serotonin, augment the production of hydrogen peroxide. We assume that this form of modulation may represent a regulatory mechanism of macrophage activation.

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.

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.

The breakdown of glycogen in the lysosomes of newborn rat hepatocytes: the effects of glucose, cyclic 3',5'-AMP and caffeine.

The effects of parenteral glucose, cyclic AMP and caffeine on the breakdown of glycogen in the lysosomes of newborn rat hepatocytes, were studied by using biochemical assays, electron microscopy and quantitative morphometry. Glucose prevented the normal postnatal increase in lysosomal volume, acid alpha 1,4 glucosidase activity and lysosomal glycogen breakdown. On the contrary, cyclic AMP and caffeine promoted this increase. There was a positive correlation between liver cyclic AMP concentration and acid glucosidase activity (R = 0.84, p < 0,001). Cyclic AMP also induced a change in the shape of lysosomes. The postulation that glucagon secreted after birth is the natural stimulus for the cyclic AMP-mediated postnatal increase in acid glucosidase activity and mobilization of the lysosomal glycogen in rat hepatocytes, is supported by these experimental findings.

A quantitative description of the insulin-induced ultrastructural changes in newborn rat hepatocytes.

The effects of insulin on the ultrastructure of newborn rat hepatocytes were systematically quantified at satisfactory statistical significance. Insulin prevented the normal postnatal increase in the total volume of lysosomes and the breakdown of glycogen inside these organelles. The lysosomal glycogen-hydrolysing enzyme, acid alpha 1,4 glucosidase was inhibited by the hormone. Insulin also prevented the normal postnatal increase in the total volume of peroxisomes, especially of the crystalloid core-devoid type. The hormone produced an increase in the area of cell membrane, due to the formation of many irregular folds of the cell surface. These results constitute good evidence for participation of lysosomes and peroxisomes in the overall glycogen degradation and or gluconeogenesis in the newborn rat hepatocytes.

Trimetazidine for prevention of hepatic injury induced by ischaemia and reperfusion in rats.

OBJECTIVE: To assess the effect of trimetazidine (an anti-anginal drug that acts as a scavenger of oxygen free radicals) in the protection of hepatocytes after a 90 minute period of warm ischaemia followed by reperfusion in rats. DESIGN: Prospective study. MATERIAL: 80 Wistar rats. INTERVENTIONS: 20 Rats were given a single dose of trimetazidine 2.5 mg/kg intravenously 30 minutes before the induction of ischaemia; 20 received the same dose intraperitoneally twice a day for five days before the experiment and one dose intravenously 30 minutes before; 20 were given a single dose of 2.5 mg/kg intravenously after reperfusion had been started; and 20 acted as controls. All rats underwent liver biopsy through a laparotomy incision on postoperative days 2, 7, and 21, and the activities of liver enzymes in their blood were measured before induction of ischaemia and two, seven, 14, and 21 days afterwards. OUTCOME MEASURES: Histological changes and serum enzyme activities. RESULTS: The amount of centrilobular necrosis of hepatocytes, and the activity of hepatic enzymes were greatest on day 2, as was the reduction in superoxide dismutase activity in the erythrocytes. A single dose of trimetazidine, whether given before or after the ischaemic episode, gave significant protection to hepatocytes, but pretreatment for five days was even more effective. CONCLUSION: Trimetazidine protected rats' livers from injury after a period of warm ischaemia and reperfusion.

Lymphocytic alveolitis and pleural calcifications in nonoccupational asbestos exposure. Protection against neoplasia?

Inhabitants of the Metsovo area in Northwest Greece (population, 4,000) have been exposed to asbestos through the use of whitewash containing tremolite. This has resulted in endemic pleural calcifications (PCs) and increased incidence of malignant pleural mesothelioma (MPM). In order to evaluate the lung response to the fiber, bronchoalveolar lavage (BAL) was performed in 25 Metsovites; 14 with PCs, three with PCs and neoplasia, five without PCs, and three without PCs but with established neoplasia. There were no differences between the four groups with regard to age or exposure. Twelve Metsovites had lymphocytic alveolitis (BAL lymphocytes > 15%). Eleven belonged to the group with PCs and one belonged to the group without PCs. None of those with neoplasia had alveolitis. The lymphocytes were mainly helper T-cells, and activation markers were more frequent among those with PCs. We have previously reported on the relative absence of PCs in Metsovites with malignant pleural mesothelioma. This observation and the results of the present study suggest that lymphocytic alveolitis correlates with pleural calcifications, whereas both are rarely present in patients with neoplasia.

An electron microscopic and biochemical study of the effects of cyclic 3', 5'-AMP, ergotamine or propranolol on the lysosomes of newborn rat hepatocytes.

The effects of cyclic 3',5'-AMP, ergotamine or propranolol on newborn rat liver were studied by using biochemical assays, electron microscopy and quantitative morphometry. Cyclic AMP enhanced the normal postnatal rise in the glycogen-hydrolysing activity of acid alpha 1, 4 glucosidase but had no effect on the maltose-hydrolysing activity of the enzyme. The results suggest that these activities may be due to different enzymes. Propranolol prevented the postnatal increase in the glycogen-hydrolysing activity of acid glucosidase and the breakdown of lysosomal glycogen, indicating that these phenomena represent beta-adrenergic functions in newborn rats. Ergotamine also inhibited the postnatal increase in this activity and the lysosomal glycogen mobilization. A reasonable explanation for these results is that ergotamine interferes with the action or formation of cyclic AMP.

Prevention of peritoneal adhesions in rats with trimetazidine.

The effect of trimetazidine (an antianginal drug that acts as a scavenger of oxygen radicals) in the prevention of peritoneal adhesions induced by complete vascular obstruction of an ileal segment for 30 minutes followed by reperfusion was investigated in rats. Group A (n = 20) acted as controls. Group B (n = 20) received trimetazidine intravenously in a dose of 2.5 mg/kg 30 minutes before the induction of ischaemia. Group C (n = 20) received the same dose of trimetazidine for 5 days before the experiment, twice a day intraperitoneally, and also intravenously 30 minutes before the induction of ischaemia. Group D (n = 20) received the same dose of trimetazidine intravenously immediately after reperfusion had started. Ten days later adhesions had developed in 90% of the animals of group A, 40% of those in group B (p less than 0.001), 5% of those in group C (p less than 0.001), and 60% of those in group D (p less than 0.05). The severity of adhesions was significantly less in the treated groups than in the control animals. Release of creatine phosphokinase during ischaemia and reperfusion significantly increase in groups A, B, and D. These results suggest that trimetazidine reduces the incidence and severity of peritoneal adhesion formation induced by ileal ischaemia and reperfusion, treatment before induction of ischaemia gave better results than treatment given afterwards.

The role of oxygen-derived free radicals in peritoneal adhesion formation induced by ileal ischaemia/reperfusion.

The effectiveness of superoxide dismutase (SOD), catalase (CAT), dimethyl sulphoxide (DMSO) and allopurinol in prevention of peritoneal adhesion formation induced by complete vascular obstruction and reperfusion of an ileal segment was investigated in rats. The ischaemic period was 30 min. Group A (n = 20) were controls, group B (n = 15) received SOD 15,000 U/kg i.v. and group C (n = 17) the same dose of CAT immediately before induction of ischaemia. In group D (n = 20) DMSO 20 mg/kg was given i.v. 5 min before ischaemia, and group E (n = 20) received allopurinol orally 50 mg/kg daily for 2 days and also 2 hours before ischaemia. Ten days later adhesions had developed in 80% of group A, 40% of group B, 47% of group C and 45% of groups D and E (p less than 0.05). The severity of the adhesions was significantly less in the pretreated groups than in the controls. Oxygen-derived free radicals may be pathogenetically important for such adhesion formation. Xanthine oxidase is the principal source of oxygen radicals after a 30-min period of complete regional intestinal ischaemia.

Ranitidine and oxygen derived free radical scavengers in haemorrhagic shock induced gastric lesions.

The role of oxygen derived free radicals in gastric lesions induced by haemorrhagic shock and the protective effect of oxygen radical scavengers, allopurinol and ranitidine, were investigated. Forty five rabbits underwent haemorrhagic shock for 30 minutes and reinfusion of shed blood. They were killed 30 minutes later. The animals were divided in five groups: A (n = 10): Control, B (n = 10): intravenous ranitidine pretreatment, C (n = 10): oral allopurinol, 24 and 2 h before surgery; D (n = 10): intravenous pretreatment with superoxide Dismutase plus catalase, E (n = 5): 60 minute haemorrhagic shock without reinfusion and treatment. Erosions and/or petechiae in all animals in Group A were observed. Three animals in group B and C and 2 in group D (p less than 0.005, p less than 0.001) had gastric lesions. The lesions in the pretreatment groups were significantly smaller than in controls. Oxygen radicals plus HCl play an important role in shock induced gastric lesions. Oxygen radical antagonists show a significant protective role.