Hepatic Lysosomes Research Articles

AYURVEDIC PROTOCOL FOR THE MANAGEMENT OF WILSON’S DISEASE – A CASE REPORT

Wilson’s disease (hepatolenticular degeneration) is a rare important autosomal recessive disorder caused by dys- function of the copper transporter ATP7B, which leads to snagging in copper transport by the hepatic lysosomes resulted in the deposition of copper in the brain, liver, kidney, or skeletal system. The symptoms are jaundice, Kayser-Fleischer rings, dysarthria, ataxia, and muscle spasticity etc. Current therapeutic modalities for the manage- ment of Wilson's disease include zinc, trientine, penicillamine etc. In Ayurvedic classics there is no exact correlation is available for this disease entity that exactly matches the feature of Wilson disease, but it can be correlated with Vatavyadhi under the Sahaja Vyadhi (Heredity) or Janamjata Vyadhi (congenital). The treatment1 mentioned for Vatavyadhi is Snehana (oleation), Mrudu Swedana (mild sedation), Anuvasana Basti (oil enema) and Niruha Basti (Decoction Enema). Material and methods: An 18-year-old male patient with Wilson disease complained of short stepping gait, persistent constipation, tremors, changed speech, generalised stiffness, and frequent eye blinking. S. Ceruloplasmin was found to be 18.33 mg/dL (usually 20 mg/dl to 40 mg/dl), S. copper was found to be within normal limits, and a slit lamp assay for the KF ring was negative. Ayurvedic therapy was used in conjunction with oral medicine throughout the treatment. Results: Clinical observations revealed that the patient's earlier symptoms had significantly improved, and he was able to carry out his usual activities with ease. Duration of treatment:three months. Conclusion: From the case study it can be concluded that the Ayurvedic medications and Panchkarma therapy in such patients may help in providing supportive care and improving the quality of life. Keywords: Vatavyadhi, Shaman Chikitsa, Bastikarma, Wilson's disease.

Artificial switching of the metabolic processing pathway of an etiologic factor, β2-microglobulin, by a “navigator” molecule

Metabolic pathways in the body are highly specific. Dysfunction of a metabolic pathway triggers the accumulation of its target substance. For example, kidney failure results in increased β2-microglobulin blood levels, causing dialysis-related amyloidosis. Previously, we proposed a novel therapeutic concept, that is a removal of an etiologic factor of metabolic disease by artificial switching of its metabolic processing pathway, and tested this concept using in cultured cells. However, the feasibility of artificial metabolic switching in vivo remained unknown. Here, we show that a newly developed “navigator” molecule changes the metabolic processing pathway of β2-microglobulin from the kidney to the liver in mouse. The artificial metabolic switching is achieved by the capture of the etiologic factor by the navigator, which then steers the etiologic factor to hepatic lysosomes via low-density lipoprotein receptors. These findings demonstrate that navigator-based artificial metabolic switching can be a therapeutic strategy for various diseases caused by metabolic disorders.

Hepatic Lysosomal iNOS Activity Impairs Autophagy in Obesity.

Background & AimsThe lysosome is an acidic organelle that is important for maintaining cellular and metabolic homeostasis in hepatocytes. Lysosomal dysfunction and chronic inflammation coexist, and both contribute to obesity-associated hepatic insulin resistance. However, in the context of obesity, the interplay between inflammatory signals and hepatic lysosomal function remains largely unknown. Inducible nitric oxide synthase (iNOS) is a hallmark for inflammation, and is activated in obesity. The aim of this study is to understand the molecular link between iNOS-mediated lysosomal nitric oxide (NO) production, hepatic lysosomal function, and autophagy in the context of obesity-associated insulin resistance.MethodsThe role of iNOS in hepatic autophagy, as related to insulin and glucose homeostasis were studied in mice with diet-induced obesity (DIO). The effects and mechanisms of iNOS-mediated lysosomal NO production on lysosomal function and hepatic autophagy were studied in primary hepatocytes as well as in a mouse model of DIO.ResultsWe demonstrate that obesity promotes iNOS localization to the lysosome and decreases levels of lysosomal arginine, resulting in an accumulation of NO in hepatic lysosomes. This lysosomal NO production is attenuated by treatment with a NO scavenger, while co-overexpression of mTOR and a lysosomal arginine transporter (SLC38A9) enhances lysosomal NO production and suppresses autophagy. In addition, we show that deletion of iNOS ameliorates lysosomal nitrosative stress in the livers of DIO mice, promotes lysosomal biogenesis by activating transcription factor EB (TFEB), and enhances lysosomal function and autophagy. Lastly, deletion of iNOS in mice with DIO improves hepatic insulin sensitivity, which is diminished by suppression of TFEB or autophagy related 7 (Atg7).ConclusionsOur studies suggest that lysosomal iNOS-mediated NO signaling disrupts hepatic lysosomal function, contributing to obesity-associated defective hepatic autophagy and insulin resistance.

Lysosomal acid lipase regulates VLDL synthesis and insulin sensitivity in mice.

Aims/hypothesisLysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (TG) within lysosomes to mobilise NEFA and cholesterol. Since LAL-deficient (Lal-/-) mice suffer from progressive loss of adipose tissue and severe accumulation of lipids in hepatic lysosomes, we hypothesised that LAL deficiency triggers alternative energy pathway(s).MethodsWe studied metabolic adaptations in Lal-/- mice.ResultsDespite loss of adipose tissue, Lal-/- mice show enhanced glucose clearance during insulin and glucose tolerance tests and have increased uptake of [3H]2-deoxy-D-glucose into skeletal muscle compared with wild-type mice. In agreement, fasted Lal-/- mice exhibit reduced glucose and glycogen levels in skeletal muscle. We observed 84% decreased plasma leptin levels and significantly reduced hepatic ATP, glucose, glycogen and glutamine concentrations in fed Lal-/- mice. Markedly reduced hepatic acyl-CoA concentrations decrease the expression of peroxisome proliferator-activated receptor α (PPARα) target genes. However, treatment of Lal-/- mice with the PPARα agonist fenofibrate further decreased plasma TG (and hepatic glucose and glycogen) concentrations in Lal-/- mice. Depletion of hepatic nuclear factor 4α and forkhead box protein a2 in fasted Lal-/- mice might be responsible for reduced expression of microsomal TG transfer protein, defective VLDL synthesis and drastically reduced plasma TG levels.Conclusions/interpretationOur findings indicate that neither activation nor inactivation of PPARα per se but rather the availability of hepatic acyl-CoA concentrations regulates VLDL synthesis and subsequent metabolic adaptations in Lal-/- mice. We conclude that decreased plasma VLDL production enhances glucose uptake into skeletal muscle to compensate for the lack of energy supply.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-3968-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Morphological alterations in the liver of yellow perch (Perca flavescens) from a biological mercury hotspot

Mercury (Hg) contamination is a global issue due to its anthropogenic release, long-range transport, and deposition in remote areas. In Kejimkujik National Park and National Historic Site, Nova Scotia, Canada, high concentrations of total mercury (THg) were found in tissues of yellow perch (Perca flavescens). The aim of this study was to evaluate a possible relationship between THg concentrations and the morphology of perch liver as a main site of metal storage and toxicity. Yellow perch were sampled from five lakes known to contain fish representing a wide range in Hg concentrations in fall 2013. The ultrastructure of hepatocytes and the distribution of Hg within the liver parenchyma were analyzed by transmission electron microscopy (TEM) and electron energy loss spectrometry (EELS). The relative area of macrophage aggregates (MAs) in the liver was determined using image analysis software and fluorescence microscopy. No relation between general health indicators (Fulton's condition index) and THg was observed. In line with this, TEM examination of the liver ultrastructure revealed no prominent pathologies related to THg accumulation. However, a morphological parameter that appeared to increase with muscle THg was the relative area of MAs in the liver. The hepatic lysosomes appeared to be enlarged in samples with the highest THg concentrations. Interestingly, EELS analysis revealed that the MAs and hepatic lysosomes contained Hg.

Protective effects of lycopene on oxidative stress, proliferation and autophagy in iron supplementation rats

Lycopene is common in diet and known for its antioxidant activities. However, the impact of lycopene on iron metabolism is poorly investigated. In this study, we hypothesize that lycopene can prevent iron-mediated oxidative stress, proliferation and autophagy in liver and use a rat model of nutritional iron supplementation to confirm its intervention in these defence mechanisms. We found that iron supplementation induced cell proliferation predominantly in non parenchymal cells compared with hepatocytes, but not apoptosis. In addition, iron was accumulated within the hepatic lysosomes where it triggered autophagy as evidenced by the formation of autophagic vesicles detected by LC3-B staining. Iron supplementation also induced morphologic alterations of the mitochondrial membranes probably due to increased lipid peroxidation as indicated by elevated iron and malondialdehyde concentrations in serum and tissues. Lycopene reduced iron-catalyzed lipid peroxidation by decreasing the malondialdehyde level in the liver and colon and enhancing the total superoxide dismutase activities in serum and tissues. The result suggest that lycopene prevents iron-induced oxidative stress, proliferation and autophagy at both biochemical and histological levels due to its potent free radical scavenging and antioxidant properties.

Lack of effect of omeprazole, a potent inhibitor of gastric (H+ + K+) ATPase, on hepatic lysosomal integrity and enzyme activity.

The substituted benzimidazole, omeprazole, is a potent inhibitor of the ATP-dependent proton pump of the parietal cell. Since there is accumulating evidence that hepatic lysosomes also possess an ATP-dependent proton pump system to maintain internal acidification, and since antibodies to the putative lysosomal proton pump protein are immunologically similar to the parietal cell (H+ + K+) ATPase, we studied the effects in rats of six days of omeprazole treatment on hepatic lysosomal function. Omeprazole, 5 mg kg-1, a dose five times the ED50 for gastric acid secretion inhibition in rats, did not alter the activity of three representative lysosomal enzymes in liver (acid phosphatase, beta-galactosidase and N-acetyl-beta-glucosaminidase) nor did it alter lysosomal enzyme latency, a measure of the integrity of the lysosomal membrane. Furthermore, bile flow and the secretion of lysosomal enzymes into bile were also unaffected by omeprazole. These data indicate that in rats short-term treatment with omeprazole, in doses that markedly inhibit gastric acid secretion, has no major biological effect on liver lysosomal integrity and lysosomal enzyme activity.

In vivo clearance and metabolism of recombinant activated factor VII (rFVIIa) and its complexes with plasma protease inhibitors in the liver

Introduction Recombinant activated factor VII (rFVIIa, NovoSeven®) is injected intravenously for the treatment of haemophilia patients with inhibitory antibodies. In plasma, rFVIIa forms complexes with protease inhibitors, primarily antithrombin III (ATIII). The liver is believed to be involved in clearance of rFVIIa, however, it is not known whether the liver is also involved for the clearance of the rFVIIa-ATIII complex. In this study, we explored the fate of intravenously injected rFVIIa from plasma to the hepatic lysosomes. Materials and methods A novel method using magnetic chromatography was used to isolate catabolic organelle (CO) fractions from mouse liver following injection of superparamagnetic dextran (SPD)-coated iron oxide particles and rFVIIa. The effect of co-circulating SPD particles on rFVIIa pharmacokinetic (PK) parameters was evaluated by ELISA. Cryo-immuno transmission electron microscopy (TEM) was used to study hepatic distribution of SPD particles and rFVIIa. The isolated hepatic CO fractions were characterized using Western Blotting (WB). Results Cryo-immuno TEM of the liver confirmed hepatic co-localisation of SPD particles and rFVIIa in identical endosomes and lysosomes of both hepatocytes and Kupffer cells. SPD particles did not affect the PK parameters of rFVIIa. WB analysis of plasma and CO fractions detected rFVIIa as the full-length protein and also in high molecular weight (HMW) complexes with ATIII and α-2 macroglobulin (α-2 M). Conclusions Following injection, both hepatocytes and Kupffer cells appeared to be involved in the hepatic clearance and metabolism of both full-length rFVIIa and rFVIIa in complex with at least two plasma protease inhibitors; ATIII and α-2 M.

Protective effects of crude garlic by reducing iron-mediated oxidative stress, proliferation and autophagy in rats

The impact of garlic, known for its antioxidant activities, on iron metabolism has been poorly investigated. The aim of this work was to study the effect of crude garlic pre-treatment on iron-mediated lipid peroxidation, proliferation and autophagy for 5 weeks. Rats were fed distilled water or garlic solution (1 g/kg body weight) by gavage for the first 3 weeks as pre-treatment and received a basal diet supplemented or not with ferrous sulfate (650 mg Fe/kg diet) for the last 2 weeks of treatment. Immunohistochemistry labeling and ultrastuctural observations were used to evaluate the iron deleterious effects in the liver. Iron supplementation induced cell proliferation predominantly in non parenchymal cells comparing to hepatocytes, but not apoptosis. In addition, iron was accumulated within the hepatic lysosomes where it triggers autophagy as evidenced by the formation of autophagic vesicles detected by LC3-II staining. It also induced morphologic alterations of the mitochondrial membranes due to increased lipid peroxidation as shown by elevated iron and malondialdehyde concentrations in serum and tissues. Garlic pre-treatment reduced iron-catalyzed lipid peroxidation by decreasing the malondialdehyde level in the liver and colon and by enhancing the status of antioxidants. In addition, garlic reduced the iron-mediated cell proliferation and autophagy by lowering iron storage in the liver and protected mitochondrial membrane. Based on these results, garlic treatment significantly prevented iron-induced oxidative stress, proliferation and autophagy at both biochemical and histological levels due to its potent free radical scavenging and antioxidant properties.

Wilson′s disease: An Indian perspective

Wilson's disease (WD) is an autosomal recessive disease involving a defect of copper transport by the hepatic lysosomes. It leads to excess copper deposition in the liver, the brain, the kidneys and the skeletal system, affecting most commonly children or young adults and running an invariably fatal course if not adequately treated by de-coppering therapy. The last century has witnessed several changes, notable among these are: Increased awareness, improved diagnostic facilities leading to earlier recognition even in the pre-symptomatic phase, clear distinction from its mimics, aggressive therapeutic approaches owing to availability of effective treatment and an overall reduction in the morbidity and mortality. It is widely acknowledged that the disease is not as rare as once believed. Sir SAK Wilson published his landmark article in 1912, but it was only in 1968 that the first patient of WD was reported from our country. Publications from India on WD have focused on phenotypic characterization, documentations of lesser recognized aspects of the disease e.g. seizures, behavior abnormality, speech and cognitive impairment, sub-clinical affection of visual pathway, heart and autonomic function and pre-symptomatic detection. Attempts have been made to understand the clinical heterogeneity of the disease through identification of biochemical and immunological markers, magnetic resonance imaging, neuropathological study and genetic analysis for novel and/or known mutations. Assessment of impairment and severity and effect of various therapeutic interventions namely zinc sulphate on the long-term outcome and quality of life have also been studied. Nevertheless, clinicians often face difficulties in long-term care of these patients. Diagnostic errors leading to delay in diagnosis and initiation of treatment are common, even in patients with positive family history. There is no consensus regarding therapeutic protocols since the use of penicillamine, once a 'gold standard' for treatment, has been debated by experts. Mortality and morbidity of this potentially treatable disease and nonavailability of medications to the poor patients remain a major area of concern.

Ultrastructural and kinetic studies of copper metabolism in Bedlington Terrier dogs.

The objectives of this study were to determine the time course of copper (Cu) absorption in the Bedlington Terrier (BT) dog after duodenal infusion of Cu and to determine the onset of Cu accumulation in BT puppies. At 80 minutes after infusion, Cu was present in the bile and liver at 26.1 and 2675 microg/g dry weight (dw), as compared with 598 and 224 microg/g dw, respectively, in the control. In the puppies, the mean liver Cu concentrations were 443 +/- 216 microng/g dw as compared with 54 +/- 34 microg/g dw in their controls. The BT puppies had large numbers of Cu granules in their hepatic lysosomes, with very few in the bile canaliculi. In contrast, the controls had few granules in their lysosomes and large numbers in their canaliculi. These results suggest that hepatic Cu accumulation occurs rapidly and in utero in the BT.

Control of radioactivity pharmacokinetics of 99mTc-HYNIC-labeled polypeptides derivatized with ternary ligand complexes.

An enhancement of the target/nontarget ratio of radioactivity levels enables reliable diagnosis and therapy using polypeptide radiopharmaceuticals in nuclear medicine. In the present study, we investigated the effects of the physicochemical properties of radiometabolites on the radioactivity pharmacokinetics after administration of 99mTc-labeled polypeptides using 6-hydrazinopyridine-3-carboxylic acid (HYNIC). Four ternary ligands (L) [3-benzoylpyridine (BP), 3-acetylpyridine (AP), 3-nicotinic acid (NIC), pyridine (PY)] with different lipophilicity were selected as coligands for the preparation of 99mTc-HYNIC-polypeptides. Each of the ternary ligands tested provided 99mTc-HYNIC-labeled galactosyl-neoalbumin (NGA) and Fab fragments of high stability with high radiochemical purity. Moreover, after administration of each 99mTc-HYNIC-labeled NGA into normal mice, the respective ternary ligand [99mTc](HYNIC-lysine)(tricine)(L) complexes were generated as final radiometabolites in the hepatic lysosome. The partition coefficients of [99mTc](HYNIC-lysine)(tricine)(BP), [99mTc](HYNIC-lysine)(tricine)(AP), [99mTc](HYNIC-lysine)(tricine)(NIC), and [99mTc](HYNIC-lysine)(tricine)(PY) were determined to be -2.21, -2.37, -2.93, and -2.73, respectively. Elimination rates of these radiometabolites from the lysosome were enhanced in the order of increasing lipophilicity of the radiometabolites. After injection of the four 99mTc-HYNIC-labeled Fab fragments into normal mice, blood clearances of radioactivity were similar while radioactivity elimination rates from the kidney were enhanced in the order of increasing lipophilicity of the radiometabolites. The present study indicated that the lipophilicity of the radiometabolites constitutes one important factor affecting their elimination rates from the tissues. Thus, as ternary ligands facilitate alteration of the physicochemical properties of radiometabolites, the use of ternary ligand complexes might be applicable for controlling the pharmacokinetics of 99mTc-labeled polypeptides.

Dissolution of copper-rich granules in hepatic lysosomes by D-penicillamine prevents the development of fulminant hepatitis in Long-Evans cinnamon rats

Background/Aim: The Long-Evans cinnamon rat has a mutation homologous to the human Wilson disease gene, leading to gross copper accumulation and the development of hepatitis. D-penicillamine, a copper-chelating drug widely and efficiently used in treating Wilson disease, has also been shown to prevent hepatitis in Long-Evans cinnamon rats. The objectives of this study were: i) to investigate the effectiveness of D-penicillamine when administered to the already affected animals, and ii) to elucidate the mechanism of action of the drug. Methods: Long-Evans cinnamon rats were divided into groups according to age and treatment with D-penicillamine. The drug was administered orally before and after the onset of hepatitis. Livers were examined by light and electron microscopy. The effect of D-penicillamine on the subcellular distribution and binding of copper was investigated in more detail. Finally, the interaction between D-penicillamine and specific hepatic copper-binding proteins was studied in vitro. Results: D-penicillamine when given to either healthy or diseased animals prevented or reversed hepatitis, respectively. The drug particularly inhibited the disease-specific accumulation of copper in lysosomes of hepatocytes, tissue macrophages and Kupffer cells. When administered to diseased animals, the drug sequestered copper particularly from insoluble lysosomal particles. According to results obtained in vitro, the mobilization of this copper is likely to proceed through the solubilization of these particles. In contrast and as supported by the in vitro data, D-penicillamine had only a minor effect on copper bound to metallothionein in the cytosol. Conclusion: Our findings on the Long-Evans cinnamon rat provide some conclusions on the mechanism of action of D-penicillamine in Wilson disease therapy. The drug prevents the formation or promotes the solubilization of copper-rich particles which occur in lysosomes of hepatocytes and Kupffer cells in the livers of patients with Wilson disease. Once chelated with D-penicillamine copper might then be excreted into urine. However, the mobilization of copper by D-penicillamine seems to be limited due to the binding of the metal to metallothionein in liver cytosol. This copper, even at relatively high concentrations, apparently may be well tolerated.

Association of copper to metallothionein in hepatic lysosomes of Long-Evans cinnamon (LEC) rats during the development of hepatitis [se e comments

The Long-Evans cinnamon (LEC) rat has a mutation homologous to the human Wilson's disease gene, leading to copper-induced hepatotoxicity. The mechanism of how excess copper damages the liver or what chemical form of copper is toxic is still unclear. In liver cytosol, copper levels were highest just before the onset of hepatitis and declined thereafter. In cytosol, total copper was bound to metallothionein (MT). Considerable amounts of both copper and iron accumulated in lysosomes with increasing age and development of liver damage. Lysosomal levels of presumably reactive non-MT-bound copper were increased. In severely affected livers, large amounts of copper were associated with insoluble material of high density which, upon ultrastructural information, was found to be derived from the lysosomes of Kupffer cells. This copper-rich material is considered to consist of polymeric degradation products of copper-MT. We suggest that chronic copper toxicity in LEC rats involves the uptake of copper-loaded MT into lysosomes, where it is incompletely degraded and polymerizes to an insoluble material containing reactive copper. This copper, together with iron, initiates lysosomal lipid peroxidation, leading to hepatocyte necrosis. Subsequent to phagocytosis by Kupffer cells, the reactive copper may amplify liver damage either directly or through stimulation of these cells.

Biliary excretion of dolichols and beta-hexosaminidase--effect of ethanol and glucagon.

Alcohol has been reported to increase the urinary excretion of dolichols, and urinary dolichols are suggested to be derived from the lysosomes of the renal cells. In the present study we examined the effects of alcohol and glucagon on the biliary excretion of dolichols in rats. Chronic ethanol treatment decreased both biliary dolichol and beta-hexosaminidase excretion. The absolute amount of dolichol excreted into the bile correlated highly significantly with the absolute amount of biliary beta-hexosaminidase. Our results indicate that biliary dolichols are--at least in part--derived from hepatic lysosomes. Decreased biliary dolichol output during chronic alcohol administration suggests that urinary and biliary dolichol excretions are regulated independently of each other.

Flow cytometric analysis of vesicular pH in rat hepatocytes after ethanol administration.

We examined the effect of ethanol administration on intravesicular pH in intact hepatocytes by applying a flow cytometric technique to detect fluorescein-isothiocyanate-dextran (FITC-dextran) in acidic vesicles. Rats were pair-fed liquid diets containing either ethanol or isocaloric carbohydrate for 1 to 5 weeks. Our study showed that ethanol administration increased the in situ pH of hepatic lysosomes by 0.15 to 0.2 pH units. This pH increase was sufficient to cause a significant reduction in lysosomal protein degradation. Long-term ethanol administration also caused a significant alkalinization of hepatic endosomes, and this increased pH was sustained over the course of vesicular acidification in hepatocytes incubated in vitro. Direct exposure of hepatocytes from rats fed control diet to either 25 mmol/L ethanol or 50 micromol/L colchicine also brought about a rapid alkalinization of acidic vesicles in a manner that resembled that seen in hepatocytes from ethanol-fed rats. These same treatments augmented the vesicular alkalinization already present in cells from ethanol-fed animals. Although ethanol administration had no effect on the content of the hepatic mannose-6-phosphate/IGFII receptor, the results indicate that sustained alkalinization of endosomes could have important functional consequences by impairing M-6-P/IGFII receptor recycling, thereby disrupting the delivery of newly synthesized hydrolases to lysosomes. This decreased complement of hydrolases within lysosomes together with alkalinization of the intralysosomal compartment would result in an overall decrease in lysosomal proteolysis.

Flow cytometric analysis of vesicular pH in rat hepatocytes after ethanol administration

We examined the effect of ethanol administration on intravesicular pH in intact hepatocytes by applying a flow cytometric technique to detect fluorescein-isothiocyanate-dextran (FITC-dextran) in acidic vesicles. Rats were pair-fed liquid diets containing either ethanol or isocaloric carbohydrate for 1 to 5 weeks. Our study showed that ethanol administration increased the in situ pH of hepatic lysosomes by 0.15 to 0.2 pH units. This pH increase was sufficient to cause a significant reduction in lysosomal protein degradation. Long-term ethanol administration also caused a significant alkalinization of hepatic endosomes, and this increased pH was sustained over the course of vesicular acidification in hepatocytes incubated in vitro. Direct exposure of hepatocytes from rats fed control diet to either 25 mmol/L ethanol or 50 micromol/L colchicine also brought about a rapid alkalinization of acidic vesicles in a manner that resembled that seen in hepatocytes from ethanol-fed rats. These same treatments augmented the vesicular alkalinization already present in cells from ethanol-fed animals. Although ethanol administration had no effect on the content of the hepatic mannose-6-phosphate/IGFII receptor, the results indicate that sustained alkalinization of endosomes could have important functional consequences by impairing M-6-P/IGFII receptor recycling, thereby disrupting the delivery of newly synthesized hydrolases to lysosomes. This decreased complement of hydrolases within lysosomes together with alkalinization of the intralysosomal compartment would result in an overall decrease in lysosomal proteolysis. (Hepatology 1997 Oct;26(4):929-34)

Ethanol consumption reduces the proteolytic capacity and protease activities of hepatic lysosomes

Chronic ethanol consumption causes decreased hepatic protein degradation, resulting in protein accumulation within hepatocytes. In this investigation, we sought to determine whether chronic ethanol feeding alters the degradative capacity and protease activities of isolated hepatic lysosomes. Male Sprague-Dawley-derived rats were fed a liquid diet containing either ethanol (36% of calories) or isocaloric maltose-dextrin for 1–5 wk. Hepatic lysosomes were isolated by differential centrifugation and purified through Percoll gradients. Lysosomes obtained from livers of ethanol-fed rats degraded both endogenous protein substrates and the exogenously added radioactive substrate, 125I-RNase A, 26–42% more slowly than lysosomes from pair fed controls. The ethanol-elicited reduction in proteolytic capacity appeared to result in part, from a deficiency of the lysosomal cathepsins B, L, and H. Compared with controls, the specific activities of these enzymes were 31–45% lower in lysosomes from ethanol-fed rats. Immunoblot analyses also revealed that the intralysosomal as well as the intracellular content of cathepsin B was significantly lower in ethanol-fed rats. In contrast, ethanol consumption did not affect the cellular quantity of cathepsin L but lowered its amount in isolated lysosomes. Our findings suggest that chronic ethanol consumption causes a deficiency in lysosomal cathepsins by altering their biosynthesis and/or their trafficking into lysosomes.

Proliferation of hepatic lysosomes and autophagic vacuoles in the Long Evans Cinnamon (LEC) rat: [formula omitted]. Department of Medicine, University of Florida, Gainesville, FL and ∗Albert Einstein College of Medicine, Bronx, NY

Proliferation of hepatic lysosomes and autophagic vacuoles in the Long Evans Cinnamon (LEC) rat: [formula omitted]. Department of Medicine, University of Florida, Gainesville, FL and ∗Albert Einstein College of Medicine, Bronx, NY

Quantitative importance of biliary excretion to the turnover of hepatic lysosomal enzymes

The turnover rate of an individual protein is a function of the rates of synthesis and loss of that protein. For most intracellular proteins, loss occurs through digestion by lysosomal or cytosolic proteases. Although a significant proportion of hepatic lysosomal enzymes is released from the hepatocyte by excretion into bile, the contribution of biliary excretion to the turnover of hepatic lysosomal enzymes has never been measured. Thus, we used in vivo pulse-labeling to determine the half-lives of two hepatic hydrolases, β-galactosidase (β-gal) and β-glucuronidase (β-glu). Each enzyme was purified by immunoisolation from hepatic lysosomes that were isolated at various times after injection of rats with 3H-labeled leucine. The decay curves for the specific radioactivities of β-gal and β-glu were used to calculate the half-lives of the proteins, which were 3.8 and 5.1 days, respectively. To determine the percent of total hepatic contents of each enzyme that was lost per day by biliary excretion, we collected bile from bile fistula rats for 24 hours and then used radioimmunoassays to quantitate the amounts of β-gal and β-glu in bile and liver samples of the same rats. We found that ∼4% of the total hepatic contents of both β-gal and β-glu was excreted into bile per day. Finally, we used these data to calculate that 31% and 41% of hepatic losses of β-gal and β-glu, respectively, were due to biliary excretion. These results suggest that extracellular release through biliary excretion is a major mechanism contributing to the turnover of lysosomal hydrolases.