Relative distribution of arylsulphatases A and B in rat liver parenchymal and other cells

The relative activities of arylsulphatases A and B were measured in rat liver parenchymal and non-parenchymal cells, in peritoneal macrophages and in a number of rat tissues. Although absolute values cannot be obtained, it was shown that the arylsulphatase B/arylsulphatase A activity ratio is much higher in non-parenchymal cells than in parenchymal cells. The ratios in adrenals, brain and testis are very similar to each other but differ from those found in spleen, kidney and liver. These ratio variations may be caused by alterations in the activity of the B enzyme rather than the A enzyme. The relatively high B enzyme/A enzyme ratios in all rat tissues explains why the method devised for the independent assay of human arylsulphatases A and B cannot be employed with rat tissues.     

Control of cell protein catabolism in rat liver. Effects of starvation and administration of cycloheximide.

1. The loss of liver protein occurring in rats starved for 24 h was largely prevented by the administration of repeated doses of cycloheximide, an inhibitor of protein synthesis. Similar effects were produced on tubulin, a 'fixed' liver protein. 2. Starvation accelerated, whereas cycloheximide markedly lowered, the rate of protein radioactivity decay after labelling with [3H]valine or [14C]bicarbonate, indicating that changes in catabolic rates played an important role in the above regulations of liver protein mass. 3. The total activity of several lysosomal hydrolases showed little change in livers of starved rats, but a marked progressive decline developed after the administration of cycloheximide, particularly in the activities of cathepsins B, D and L as well as acid ribonuclease. There was no evidence that these changes might be due to endogenous inhibitors (at least for cathepsin B activity, which fell to less than 30% of the control values) or enzyme leakage into the bloodstream; rather, plasma beta-galactosidase and beta-N-acetylglucosaminidase activities fell progressively during the cycloheximide treatment. 4. Endogenous proteolytic rates, measured in vitro by incubating subcellular preparations from livers prelabelled in vivo with [3H]valine, were markedly decreased in cycloheximide-treated animals. 5. The osmotic fragility of hepatic lysosomes, appreciably enhanced in starved animals, after cycloheximide treatment was found to be even lower than in fed controls. 6. The present data are consistent with the view that in starved animals the loss of liver protein is mostly accounted for by increased breakdown, due, in part at least, to enhanced autophagocytosis. 7. Cycloheximide largely counteracted these effects of starvation, altering the liver from being 'poised' in a proteolytic direction to a protein-sparing condition. The present data suggest that, besides suppression of the autophagic processes, a decrease in the lysosomal proteolytic enzyme system may also play a role in this regulation, and they seem to provide further circumstantial evidence for the existence of co-ordinating mechanisms between protein synthesis and degradation.     

Enzymic studies of sulphatases in tissues of the normal human and in metachromatic leukodystrophy with multiple sulphatase deficiencies: arylsulphatases A, B and C, cerebroside sulphatase, psychosine sulphatase and steroid sulphatases

Abstract— Several sulphatases (arylsulphatases A, B and C, cholesterol sulphatase, dehydroepiandroster-one sulphatase, cerebroside sulphatase and psychosine sulphatase) were deficient in various tissues from two patients with a variant form of metachromatic leukodystrophy. Deficient activities of cerebroside sulphatase and psychosine sulphatase, using physiological substrates, in tissues from metachromatic leukodystrophy with multiple sulphatase deficiencies provided another example that these enzymes may be identical to arylsulphatase A. β-Galactosidase activity was reduced to about 30-50 per cent of normal in brain and liver. Other lysosomal enzyme activities were found to be normal or elevated five to eight times. Arylsulphatase B isolated from the liver of one patient was abnormal, with respect to pi (70) and enzyme kinetics. In mixing experiments with normal enzymes the reduced activities of arylsulphatases A. B and C, cerebroside sulphatase and steroid sulphatases were shown not to be due to the presence of endogenous inhibitors. No arylsulphatase A or B activity in the brain specimen from the patient with multiple sulphatase deficiencies could be detected on isoelectric focussing. In normal brain tissue arylsulphatase A had a pi of 4-6-4-8 while arylsulphatase B had a pi of 7-8 and 8-1. When 4-methylumbelliferyl sulphate was used as a substrate the elution patterns of normal brain and liver arylsulphatase B were more heterogeneous and showed more variation than that when p-nitrocatechol sulphate was used. Arylsulphatase C and steroid sulphatases (cholesterol sulphatase, dehydroepiandrosterone sulphatase and oes-trone sulphatase I were solubilized by the addition of lysolecithin and Triton X-100 and subjected to isoelectric focussing. The pi of cholesterol sulphatase, oestrone sulphatase and arylsulphatase C was 6-8, and the elution patterns of the activities of these enzymes were similar. The pattern of dehydroepiandrosterone sulphatase was more heterogeneous and two major peaks were observed at pi 6 5 and 70. Residual enzyme activities of arylsulphatase C and steroid sulphatases from the brain of the patient with multiple sulphatase activities were not detectable by isoelectric focussing. Simultaneous deficiencies of arylsulphatase C and steroid sulphatases plus isoelectric focussing findings in tissues suggest that these enzymes are closely related in regard to their function. The nature of the genetic defect in metachromatic leukodystrophy with multiple sulphatase deficiencies is discussed.     

Effects of acute and chronic administration of ethanol on rat brain arylsulphatase A and B

Acute (4g/kg i.p.) and chronic (Sustacal^TM diet containing 10% ethanol for 20 days) administration of ethanol to male Sprague-Dawley rats produced no change in the content or enzyme activity of brain arylsulphatase A. In contrast to the lack of effect on arylsulphatase A, the acute and chronic administration of ethanol resulted in an increase in the activity of brain arylsulphatase B (15.8% and 18.4%, respectively). However, the enhancement of the activity of arylsulphatase B was observed only in the brain homogenates which were subjected to osmotic shock. No enhancement of the arylsulphatase B activity was found in the supernatant soluble fraction after the acute and chronic administration of ethanol. Furthermore, acute and chronic ethanol administration did not alter the activities of arylsulphatase A and B in microsomes which have been suggested as sites of the synthesis of lysosomal hydrolases. In addition, 80 mM ethanol, in vitro, did not affect the activity of arylsulphatase A and B. The results of the present study suggest that the acute or chronic administration of ethanol might enhance the activity of lysosomal membrane bound arylsulphatase B via altering the lipid metabolism of lysosomal membranes.     

Purification of soluble arylsulphatases from ox brain

1. Two soluble arylsulphatases (A and B) have been extracted from ox brain by a modified Albers autolysis method and purified by acetone and ammonium sulphate precipitation and dialysis. 2. A 1600-fold purification was achieved with arylsulphatase A and 320-fold purification with arylsulphatase B. 3. The specific activity of arylsulphatase A was 266000 4-nitrocatechol units/mg. of protein N, and that of arylsulphatase B was 64600units/mg. of protein N. 4. Arylsulphatase A seems to be electrophoretically homogeneous. 5. With 3mm-dipotassium 2-hydroxy-5-nitrophenyl sulphate as substrate the optimum pH for the activity of arylsulphatase A was 4.7, and for arylsulphatase B it was 6.1 with a 60mm solution of the same substrate.     

Ketone-body metabolism after partial hepatectomy in the rat.

Fed or 24 h-starved rats were subjected to two-thirds partial hepatectomy or sham-operation and subsequently starved for 4, 14 or 24 h. Despite high plasma fatty acid concentrations, the partially hepatectomized rats failed to respond to post-operative starvation with increased blood and liver ketone-body concentrations or to maintain the high ketone-body concentrations associated with pre-operative starvation. Hypoglycaemia and hyperlactaemia were observed within 30 min of functional hepatectomy, but not partial hepatectomy, of 24 h-starved rats, and, even after a further 24 h starvation of partially hepatectomized rats, blood glucose concentrations were only slightly decreased. The results are discussed with reference to fat oxidation and gluconeogenesis in the liver remaining after partial hepatectomy.     

Arylsulphatases in the rabbit oviduct: postovulatory changes tested by histochemical and biochemical procedures

Summary A histochemical and biochemical study of the activity of arylsulphatases A and B was carried out on the oviduct of female rabbits during the first days after mating. The histochemical results demonstrated that the ampullary and the isthmic epithelial cells have a positive reaction to the sulphatases during the whole of the postovulatory period tested. The enzymatic activity is mainly localized in the basal cellular cytoplasm. The biochemical results confirmed that both arylsulphatase A and B are active. Arylsulphatase A activity is more intense in the ampulla than in the isthmus and it increases during the whole of the postovulatory period; in the isthmus the activity increases up to 72 h, thereafter decreasing again. The arylsulphatase B activity is always lower than arylsulphatase A activity; maximum activity is reached between 66 to 72 h after mating. The arylsulphatase B is relatively higher in the ampulla than in the isthmus. The biological role of these enzymes is discussed in relation to the regulation of the sulphated glycoconjugates.     

Starvation-induced autophagocytosis paradoxically decreases the susceptibility to oxidative stress of the extremely oxidative stress-sensitive NIT insulinoma cells

Summary

Glucose and amino acid starvation of cells in culture generally enhances their sensitivity to oxidative stress. This is explained by compensatory autophagocytosis, which results in increased amounts of lysosomal low-molecular-weight, redox-active iron, due to the degradation of metallo-proteins, with a potential increase in iron-catalyzed, intralysosomal oxidative reactions. Such reactions diminish the stability of lysosomal membranes, with resultant leakage of hydrolytic enzymes into the cytosol and ensuing cellular degeneration, often of apoptotic type. However, starvation of NIT insulinoma cells, which are normally remarkably sensitive to oxidative stress, actually attenuated the sensitivity to such stress. We found that starved NIT cells rapidly synthesized ferritin. Moreover, ferritin was found to be autophagocytosed, and the lysosomes were stabilized, as assayed by the acridine orange relocation test. We hypothesize that compensatory autophagocytosis during starvation increases the cytosolic pool of redox-active iron, as a reflection of enhanced transportation of low-molecular-weight iron from autophagic lysosomes to the cytosol, resulting in ferritin induction. The newly formed ferritin would, in turn, become autophagocytosed and bind redox-active lysosomal iron in a non-redox-active form. We also suggest that the proposed mechanism may be a way for oxidative stress-sensitive cells to compensate partly for their failing capacity to degrade hydrogen peroxide before it leaks into the acidic vacuolar apparatus and induces intralysosomal oxidative stress. The insulin-producing beta cell may belong to this type of cells.     

Effect of starvation and refeeding on autophagy and heterophagy in rat liver

Increase in the density of liver lysosomes after leupeptin administration was marked in starved rats but only slight in starved-refed rats. The levels of several intracellular enzymes in the liver lysosome fraction purified from leupeptin-treated rats were about 10 to 30 times more in starved rats than in refed rats. However, there was no difference between the intralysosomal levels of endocytosed FITC-labeled asialofetuin in starved and refed rats, indicating that refeeding after starvation markedly suppressed autophagy but not heterophagy in vivo. Immunohistochemical studies with cathepsin B and asialofetuin Fab'-peroxidase conjugates showed that refeeding after starvation markedly altered the cellular distribution of cathepsin B in the liver, resulting in a linear arrangement of the enzyme only on the periphery of hepatocytes. In contrast, endocytosed asialofetuin was found only in the periphery of hepatocytes of both starved and starved-refed rats. These results indicate that autophagy and heterophagy are regulated by different mechanisms in vivo.     

Effects of starvation and refeeding a high carbohydrate diet on the intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in the liver of male and female rats

Phosphoenolpyruvate carboxykinase activity in rat liver was shown to be heterotopically distributed within the acinus under varying feeding conditions. Highest values of PEPCK activity were found in the periportal zone of the acinus from where it decreased continuously towards the perivenous zone. 84 h of starvation resulted in an increase of activity, which was most prominent in the perivenous zone, but nevertheless resulted in a steeper gradient. Refeeding of starved rats with a high carbohydrate diet for 6 nights led to a decrease in PEPCK activity which was most prominent in the periportal zone, but almost negligible in the perivenous zone, resulting in a further change in the activity gradient. Sex-dependent differences for total PEPCK activity were found i) in controls, where the activity was lower in females, ii) after starvation, where the induction was much higher in females, and iii) after refeeding of starved rats, where the activity in females remained higher compared to that of the controls. Differences in the intra-acinar localization of the activity in dependence of the sex were registrated in the control group and in starved rats. Livers from female rats contained a higher periportal/perivenous ratio compared to males. In starved and starved and refed animals the periportal/perivenous ratios were almost the same in both sexes.     

The effect of starvation on the incorporation of palmitate into glycerides and phospholipids of rat liver homogenates

1. Glyceride biosynthesis from glycerol phosphate and [1-(14)C]palmitate was studied in liver homogenates of rats that were fed ad libitum or starved for 36-40hr. The changes in enzyme activity were related to total DNA content or total liver homogenate as these were found to be equivalent and to be the most meaningful parameters. 2. In liver homogenates from fed rats, labelled palmitate was incorporated mainly into phosphatidate (58% of the total incorporation into lipids), diglycerides (25%) and triglycerides (16%), whereas monoglycerides, cholesterol esters and phospholipids other than phosphatidate were labelled only to a small extent. Addition of particle-free supernatant to full homogenates increased the total incorporation of palmitate by 45% and the pattern of incorporation altered to 53% incorporated into triglycerides, 24% into diglycerides and 17% into phosphatidate. This result suggested that, in liver homogenates, phosphatidate phosphohydrolase (EC 3.1.3.4) may be rate-limiting in the biosynthesis of glycerides via the glycerol phosphate pathway. 3. Upon starvation, the amount of palmitate incorporated per liver into total phospholipids plus glycerides was decreased to between 68% and 75% of that observed with fed animals. In homogenates from fed animals 41-44% of the labelled phospholipids plus glycerides was in glycerides; this value increased to between 63% and 75% with starved rats. Of the palmitate incorporated into total phospholipids, between 85% and 86% was found in phosphatidate, independent of the nutritional state of the animal. The ratio of palmitate incorporated into triglycerides/diglycerides rose from 0.7, obtained with fed rats, to 1.0 with starved animals. 4. These results indicate that starvation caused a decrease in the activity (per total liver) of acyl-CoA-glycerol phosphate acyltransferase(s) (EC 2.3.1.15) and an increase in the activity of acyl-CoA-diglyceride acyltransferase (EC 2.3.1.20). The largest change, however, seemed to be related to the increased activity of the phosphatidate phosphohydrolase in the particle-free supernatant. 5. The latter enzyme was assayed in the particle-free supernatant with membrane-bound phosphatidate as substrate. In starvation, the activity per total liver was increased to between 130% and 190% and the specific activity to between 180% and 320% of the values for fed rats.     

Effects of starvation and refeeding a high carbohydrate diet on the intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in the liver of male and female rats

Summary Phosphoenolpyruvate carboxykinase activity in rat liver was shown to be heterotopically distributed within the acinus under varying feeding conditions. Highest values of PEPCK activity were found in the periportal zone of the acinus from where it decreased continuously towards the perivenous zone. 84 h of starvation resulted in an increase of activity, which was most prominent in the perivenous zone, but nevertheless resulted in a steeper gradient. Refeeding of starved rats with a high carbohydrate diet for 6 nights led to a decrease in PEPCK activity which was most prominent in the periportal zone, but almost negligible in the perivenous zone, resulting in a further change in the activity gradient.Sex-dependent differences for total PEPCK activity were found i) in controls, where the activity was lower in females, ii) after starvation, where the induction was much higher in females, and iii) after refeeding of starved rats, where the activity in females remained higher compared to that of the controls. Differences in the intra-acinar localization of the activity in dependence of the sex were registrated in the control group and in starved rats. Livers from female rats contained a higher periportal/perivenous ratio compared to males. In starved and starved and refed animals the periportal/perivenous ratios were almost the same in both sexes.     

Increases in hepatic fructose-2,6-bisphosphate level and fructose-6-phosphate,2-kinase activity in rats with ventromedial lesions of the hypothalamus

To investigate altered fructose-2,6-bisphosphate (fructose-2,6-P2) metabolism, we measured fructose-2,6-P2 levels and fructose-6-phosphate,2-kinase (fructose-6-P,2-kinase) activities in various tissues, including liver, kidney, heart, and skeletal muscle, of ventromedial hypothalamus (VMH)-lesioned rats during feeding and starvation. The plasma insulin level was 6 times or more higher in these rats than in the controls. The fructose-2,6-P2 level in liver was much greater in VMH-lesioned rats than in the controls: 15.1 +/- 2.2 nmol/g tissue versus 7.7 +/- 0.7 in the fed state, 5.3 +/- 1.1 versus 1.6 +/- 0.4 in the starved state. In kidney, heart, and skeletal muscle, fructose-2,6-P2 levels were not different between the two animal groups. The activity of hepatic fructose-6-P,2-kinase remained high after 20 h of starvation in VMH-lesioned rats, whereas it was decreased markedly in the controls. The hepatic concentration of fructose-6-phosphate was also high in VMH-lesioned rats. Both fructose-6-P,2-kinase activity and fructose-6-phosphate concentration in the liver of starved VMH-lesioned rats were comparable to those of control rats in fed conditions. These results indicate that the alteration of fructose-2,6-P2 metabolism is characteristic of liver in VMH-lesioned rats, and that the increase in hepatic fructose-2,6-P2 may activate hepatic glycolysis not only during feeding but also during starvation, leading to the enhanced lipogenesis in these obese rats.     

Evidence that lysosomes are not involved in the degradation of myofibrillar proteins in rat skeletal muscle.

To examine the role of lysosomes in the degradation of skeletal-muscle myofibrillar proteins, we measured the release of N tau-methylhistidine from perfused muscle of starved and fed rats in the presence or absence of agents that inhibit lysosomal proteinase activity. After 1 day of starvation, the release of N tau-methylhistidine by perfused muscle of 4-, 8- and 24-week-old rats increased by 322, 159 and 134% respectively. On the other hand, total protein breakdown, assessed by tyrosine release, increased by 62, 20 and 20% respectively. Inhibitors of lysosomal proteinases as well as high concentrations of insulin or amino acids failed to diminish the release of N tau-methylhistidine by perfused muscle of starved and fed rats, despite a 25-35% inhibition of total protein breakdown. The data strongly suggest that the complete breakdown of myofibrillar proteins occurs via a non-lysosomal pathway. They also suggest that total proteolysis, which primarily reflects non-myofibrillar protein breakdown, occurs at least in part within lysosomes.     

The activity of glycogen synthase phosphatase limits hepatic glycogen deposition in the adrenalectomized starved rat.

Hepatocytes from adrenalectomized 48 h-starved rats responded to increasing glucose concentrations with a progressively more complete inactivation of phosphorylase. Yet no activation of glycogen synthase occurred, even in a K+-rich medium. Protein phosphatase activities in crude liver preparations were assayed with purified substrates. Adrenalectomy plus starvation decreased synthase phosphatase activity by about 90%, but hardly affected phosphorylase phosphatase activity. Synthase b present in liver extracts from adrenalectomized starved rats was rapidly and completely converted into the a form on addition of liver extract from a normal fed rat. Glycogen synthesis can be slowly re-induced by administration of either glucose or cortisol to the deficient rats. In these conditions there was a close correspondence between the initial recovery of synthase phosphatase activity and the amount of synthase a present in the liver. The latter parameter was strictly correlated with the measured rate of glycogen synthesis in vivo. The decreased activity of synthase phosphatase emerges thus as the single factor that limits hepatic glycogen deposition in the adrenalectomized starved rat.     

Fluctuations in arylsulphatase activity in the rabbit endometrium during the sexual cycle

Summary Histochemical and biochemical studies were performed to verify the presence of arylsulphatase A (ASA) and B (ASB) in the rabbit uterus. Fluctuations in the activity of these sulphatases during the sexual cycle were also studied. Some structural and functional properties of purified ASA were determined. The results indicate that arylsulphatases are active in the endometrium during both the estrogenic and progesteronic phases. The activity of ASA was much more intense than that of ASB; it increased during estrus and decreased during the post-ovulatory phase. ASB activity, however, decreased during estrus and increased during the post-ovulatory phase. The significance of these fluctuations is discussed in relation to the action of sexual hormones and physiological substrates of arylsulphatases.     

Vinblastine-induced autophagocytosis: effects on liver glycogen

The possible similarities of the mechanism by which vinblastine induces autophagocytosis in liver were compared with the known effects of glucagon in glucagon-induced autophagocytosis. A single intraperitoneal injection of vinblastine produced a wave of autophagocytosis in less than 0.5 h in mouse hepatocytes. Liver glycogen content decreases simultaneously and blood glucose first increased and then decreased below control values. Both liver cAMP concentration and the activity of glycogen phosphorylase remained unchanged. These findings provide evidence that the induction of autophagocytosis after vinblastine injection is not mediated by cAMP. The increased degradation of glycogen may occur in the lysosomal system by means of increased autophagocytosis.     

Fluctuations in arylsulphatase activity in the rabbit endometrium during the sexual cycle

Histochemical and biochemical studies were performed to verify the presence of arylsulphatase A (ASA) and B (ASB) in the rabbit uterus. Fluctuations in the activity of these sulphatases during the sexual cycle were also studied. Some structural and functional properties of purified ASA were determined. The results indicate that arylsulphatases are active in the endometrium during both the estrogenic and progesteronic phases. The activity of ASA was much more intense than that of ASB; it increased during estrus and decreased during the post-ovulatory phase. ASB activity, however, decreased during estrus and increased during the post-ovulatory phase. The significance of these fluctuations is discussed in relation to the action of sexual hormones and physiological substrates of arylsulphatases.     

Synthesis and degradation of phosphoenolpyruvate carboxylase in rat liver and adipose tissue. Changes during a starvation-re-feeding cycle

A specific antibody against liver cytosol phosphoenolpyruvate carboxylase (EC 4.1.1.32) was used to isolate the enzyme from liver and adipose tissue. With this technique we have shown that phosphoenolpyruvate carboxylase synthesis in starved rats accounts for 3% of the total synthesis of cytosol protein in each tissue. Re-feeding starved animals decreases this relative rate of phosphoenolpyruvate carboxylase synthesis to 0.2% and 1% respectively in liver and adipose tissue, and the activity of the enzyme in each tissue is decreased to 25% of the starvation value. An additional starvation period is accompanied by an increased rate of enzyme synthesis, but the response to starvation is considerably slower than that caused by re-feeding. The degradation rate of phosphoenolpyruvate carboxylase is also subject to regulation. Thus re-feeding starved animals decreases the half-life of the enzyme in liver from 13h to 5.2h, but the rapid rate of degradation is maintained at least during the first 20h of subsequent starvation. Only slight changes in the degradation rate of phosphoenolpyruvate carboxylase are found in adipose tissue. We conclude that the large alterations in the rate of enzyme synthesis during a starvation–re-feeding cycle are the major cause of fluctuations in activity.     

Influence of starvation,Triton WR-1339 and [131I]-human serum albumin on rat liver lysosomes

The response of rat liver lysosomes to starvation and administration of lysosomotropic agentsviz. Triton WR-1339 and [¹³¹I]-human serum albumin, was assessed in terms of their distribution pattern after isopycnic sucrose density gradient centrifugation. Starvation induced changes in lysosomes appeared to be similar to that produced by the detergent uptake. Both the treatments caused a distinct decline in the equilibration densities of the organelles. On the other hand, injected labelled protein failed to comigrate with the lysosomal markers in starved as well as Triton treated rats and conspicuously remained in a region of high specific gravity in the gradient. These findings indicate retarded fusion between secondary lysosomes and [¹³¹I]-human serum albumin containing phagosomes in the livers of rats subjected to starvation or detergent treatment