scholarly journals Phosphoinositide 3-kinase regulates maturation of lysosomes in rat hepatocytes

2003 ◽  
Vol 372 (3) ◽  
pp. 861-869 ◽  
Author(s):  
Seyed Ali MOUSAVI ◽  
Andreas BRECH ◽  
Trond BERG ◽  
Rune KJEKEN
Keyword(s):  
Intracellular Transport ◽  
Degradation Products ◽  
Buoyant Density ◽  
Rat Hepatocytes ◽  
Amino Acid Mixture ◽  
Endocytic Pathway ◽  
Acid Mixture ◽  
Density Distributions ◽  
Late Endosomes ◽  
Phosphoinositide 3 Kinase

To obtain information about the role of phosphoinositide 3-kinase (PI3K) in the endocytic pathway in hepatocytes, the uptake and intracellular transport of asialo-orosomucoid (ASOR) was followed in cells treated with wortmannin or LY294002. The two inhibitors, at concentrations known to inhibit the enzyme, did not affect internalization or the number of surface asialoglycoprotein receptors, but they caused a paradoxical increase (approx. 50% above control values) in the degradation of ASOR labelled with [125I]tyramine cellobiose ([125I]TC). Wortmannin or LY204002 inhibited the autophagic sequestration of lactate dehydrogenase very effectively, and the enhanced degradation of [125I]TC-ASOR could be an indirect effect of reduced autophagy, as an amino acid mixture known to inhibit autophagy also caused increased degradation of [125I]TC-ASOR, and its effect was not additive to that of wortmannin or LY294002. Wortmannin or LY294002 had pronounced effects on the late parts of the endocytic pathway in the hepatocytes: first, dense lysosomes disappeared and were replaced by swollen vesicles; secondly, degradation of [125I]TC-ASOR took place in an organelle of lower buoyant density (in a sucrose gradient) than the bulk of lysosomes (identified in the gradient by lysosomal marker enzymes). With increasing length of incubation with wortmannin or LY294002, the density distributions of the lysosomal markers also shifted to lower density and gradually approached that of the labelled degradation products. The labelled degradation products formed from [125I]TC-labelled proteins were trapped at the site of formation, because they did not penetrate the vesicle membranes. The results obtained indicate that internalization and intracellular transport of ASOR to lysomes may take place in the absence of PI3K activity in rat hepatocytes. On the other hand, fusion of late endosomes with lysosomes seems to produce ‘hybrid organelles’ (active lysosomes) that are unable to mature into dense lysosomes.

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

1985 ◽  
Vol 232 (3) ◽  
pp. 773-780 ◽  
Author(s):  
P B Gordon ◽  
H Tolleshaug ◽  
P O Seglen
Keyword(s):  
Rat Hepatocytes ◽  
Amino Acid Mixture ◽  
Sugar Uptake ◽  
Acid Mixture ◽  
Detectable Effect ◽  
Isolated Rat Hepatocytes ◽  
Assay Procedure ◽  
Low Concentrations ◽  
Extraction Step ◽  
Isolated Rat

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.

scholarly journals Use of glycyl-l-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles

1994 ◽  
Vol 300 (1) ◽  
pp. 229-236 ◽  
Author(s):  
T O Berg ◽  
P E Strømhaug ◽  
T Løvdal ◽  
P O Seglen ◽  
T Berg
Keyword(s):  
Acid Phosphatase ◽  
Degradation Products ◽  
Rat Hepatocytes ◽  
Endocytic Pathway ◽  
Marker Enzyme ◽  
Major Peak ◽  
Isolated Rat Hepatocytes ◽  
Cathepsin C ◽  
Minor Peak ◽  
A Minor

Lysosome-disrupting enzyme substrates have been used to distinguish between lysosomal and prelysosomal compartments along the endocytic pathway in isolated rat hepatocytes. The cells were incubated for various periods of time with 125I-labelled tyramine cellobiose (125I-TC) covalently coupled to asialoorosomucoid (AOM) (125I-TC-AOM); this molecule is internalized by receptor-mediated endocytosis and degraded in lysosomes, where the degradation products (acid-soluble, radio-labelled short peptides) accumulate, Glycyl-L-phenylalanine 2-naphthylamide (GPN) and methionine O-methyl ester (MOM), which are hydrolysed by lysosomal cathepsin C and a lysosomal esterase respectively, both diffused into hepatocytic lysosomes after electrodisruption of the cells. Intralysosomal accumulation of the hydrolysis products (amino acids) of these substrates caused osmotic lysis of more than 90% of the lysosomes, as measured by the release of acid-soluble radioactivity derived from 125I-TC-AOM degradation. The acid-soluble radioactivity coincided in sucrose-density gradients with a major peak of the lysosomal marker enzyme acid phosphatase at 1.18 g/ml; in addition a minor, presumably endosomal, acid phosphatase peak was observed around 1.14 g/ml. The major peak of acid phosphatase was almost completely released by GPN (and by MOM), while the minor peak seemed unaffected by GPN. Acid-insoluble radioactivity, presumably in endosomes, banded (after 1 h of 125I-TC-AOM uptake) as a major peak at 1.14 and a minor peak at 1.18 g/ml in sucrose gradients, and was not significantly released by GPN. GPN thus appears to be an excellent tool by which to distinguish between endosomes and lysosomes. MOM, on the other hand, released some radioactivity and acid phosphatase from endosomes as well as from lysosomes.

scholarly journals Amino acid control of autophagic sequestration and protein degradation in isolated rat hepatocytes.

1984 ◽  
Vol 99 (2) ◽  
pp. 435-444 ◽  
Author(s):  
P O Seglen ◽  
P B Gordon
Keyword(s):  
Amino Acid ◽  
Protein Degradation ◽  
Rat Hepatocytes ◽  
Amino Acid Mixture ◽  
Regulatory Function ◽  
Acid Mixture ◽  
Cellular Functions ◽  
Isolated Rat Hepatocytes ◽  
Fluid Uptake ◽  
Isolated Rat

Sequestration of the inert cytosolic marker [14C]sucrose by sedimentable organelles was measured in isolated rat hepatocytes made transiently permeable to sucrose by means of electropermeabilization. Lysosomal integrity, protein degradation, autophagic sequestration, and other cellular functions were not significantly impaired by the electric treatment. Hepatocytes sequestered sucrose at an initial rate of approximately 10%/h, which is threefold higher than the estimated rate of autophagic-lysosomal protein degradation. Almost one-third would appear to represent mitochondrial fluid uptake; the rest was nearly completely and specifically inhibited by 3-methyladenine (3MA) and can be regarded as autophagic sequestration. A complete amino acid mixture was somewhat less inhibitory than 3MA, and partially antagonized the effect of the latter. This paradoxical effect, taken together with the high sequestration rate, may suggest heterogeneity as well as selectivity in autophagic sequestration. There was no detectable recycling of sequestered [14C]sucrose between organelles and cytosol. Studies of individual amino acids revealed histidine as the most effective sequestration inhibitor. Leucine may have a regulatory function, as indicated by its unique additive/synergistic effect, and a combination of Leu + His was as effective as the complete amino acid mixture. Asparagine inhibited sequestration only 20%, i.e., its very strong effect on overall (long-lived) protein degradation must partially be due to post-sequestrational inhibition. The lysosomal (amine-sensitive) degradation of short-lived protein was incompletely inhibited by 3MA, indicating a contribution from nonautophagic processes like crinophagy and endocytic membrane influx. The ability of an amino acid mixture to specifically antagonize the inhibition of short-lived protein degradation by AsN + GIN (but not by 3MA) may suggest complex amino acid interactions at the level of fusion between lysosomes and other vesicles in addition to the equally complex interactions at the level of autophagic sequestration.

Isolation and characterization of early endosomes, late endosomes and terminal lysosomes: their role in protein degradation

1996 ◽  
Vol 109 (12) ◽  
pp. 2905-2914 ◽  
Author(s):  
T.E. Tjelle ◽  
A. Brech ◽  
L.K. Juvet ◽  
G. Griffiths ◽  
T. Berg
Keyword(s):  
Lysosomal Enzymes ◽  
Degradation Products ◽  
Mannose Receptor ◽  
Subcellular Fractionation ◽  
Endocytic Pathway ◽  
Acid Hydrolases ◽  
Main Site ◽  
Isolation And Characterization ◽  
Late Endosomes ◽  
Early Endosomes

Although endosomal proteolysis has been reported (e.g. for peptide hormones and lysosomal enzymes), lysosomes are believed to be the main site of degradation in the endocytic pathway. We have studied the separate roles of lysosomes and prelysosomal endocytic organelles in the degradation of ovalbumin in J774 cells. The ovalbumin was labelled with 125I-tyramine cellobiose (125I-TC-ova). The labelled degradation products formed from this probe are trapped at the site of formation. To separate lysosomes efficiently from prelysosomal endocytic organelles we allowed the cells to endocytose a pulse of colloidal gold particles complexed with ovalbumin. By combining this density shift technique with subcellular fractionation of a postnuclear supernatant in Percoll gradients we could isolate three fractions that were sequentially involved in the endocytic pathway: a light Percoll fraction, a dense Percoll fraction and a gold fraction. The light Percoll fraction contained early endosomes since it was transferrin positive and received endocytic markers such as ovalbumin and horseradish peroxidase (HRP) early (< 5 minutes) after internalization. The dense Percoll fraction was transferrin negative, rab7 positive and received endocytic markers after 10–15 minutes of internalization. The gold-filled fraction was negative for both transferrin and rab7 but highly enriched in the lysosomal enzyme beta-hexosaminidase and was therefore defined as a lysosome. To study the role of endosomes and lysosomes in the degradation of endocytosed material we allowed the cells to take up (via the mannose receptor) 125I-TC-ova. It was found that the main degradation of 125I-TC-ova (measured as acid soluble radioactivity trapped in the organelle) took place in the late endosomes (and not in the lysosomes containing the bulk of the lysosomal enzymes). Our data therefore suggest that the late endosomes operate as an early lysosomal compartment. The terminal lysosomes may serve as storage bodies for acid hydrolases that may be called upon when needed (for instance during phagocytosis).

scholarly journals Effects of amino acids, ammonia and leupeptin on protein synthesis and degradation in isolated rat hepatocytes

1978 ◽  
Vol 174 (2) ◽  
pp. 469-474 ◽  
Author(s):  
P O Seglen
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Protein Degradation ◽  
Specific Radioactivity ◽  
Rat Hepatocytes ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Isolated Rat Hepatocytes ◽  
Lysosomal Protein ◽  
Isolated Rat

Protein synthesis in isolated rat hepatocytes, as measured by the incorporation of [14C]-valine at constant specific radioactivity, proceeded at a rate of 0.3-0.5%/h in an unsupplemented medium, i.e. only about one-tenth the rate of protein degradation (4%/h). Leupeptin, which inhibits lysosomal protein degradation (previously found to be 75% of the total degradation in hepatocytes), had no effect on protein synthesis, showing that endogenous protein degradation supplied amino acids in excess of the substrate requirements for protein synthesis. The inhibition of protein synthesis by NH4Cl (another inhibitor of lysosomal protein degradation) as well as the stimulation by a physiological amino acid mixture must therefore represent indirect effects, either on general energy metabolism, or on unknown regulatory processes.

An open-label, single-center pilot study to test the effects of an amino acid mixture in older patients admitted to internal medicine wards

Nutrition ◽  
2020 ◽  
Vol 69 ◽  
pp. 110588 ◽  
Author(s):  
Francesco Bellanti ◽  
Aurelio Lo Buglio ◽  
Elena Di Stasio ◽  
Giorgia di Bello ◽  
Rosanna Tamborra ◽  
...  
Keyword(s):  
Internal Medicine ◽  
Amino Acid ◽  
Pilot Study ◽  
Older Patients ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Single Center ◽  
Open Label ◽  
Internal Medicine Wards

Choosing A Balanced Amino Acid Mixture

1970 ◽  
Vol 100 (3) ◽  
pp. 380-380 ◽  
Author(s):  
Hans Fisher
Keyword(s):  
Amino Acid ◽  
Amino Acid Mixture ◽  
Acid Mixture

scholarly journals Effect of co-ingestion of amino acids with rice on glycaemic and insulinaemic response

2015 ◽  
Vol 114 (11) ◽  
pp. 1845-1851 ◽  
Author(s):  
Yean Yean Soong ◽  
Joseph Lim ◽  
Lijuan Sun ◽  
Christiani Jeyakumar Henry
Keyword(s):  
Amino Acids ◽  
Blood Glucose ◽  
Amino Acid ◽  
Glycaemic Index ◽  
Amino Acid Mixture ◽  
Postprandial Blood Glucose ◽  
Acid Mixture ◽  
White Rice ◽  
Amino Acid Mixtures

AbstractConsumption of high glycaemic index (GI) and glycaemic response (GR) food such as white rice has been implicated in the development of type 2 diabetes. Previous studies have reported the ability of individual amino acids to reduce GR of carbohydrate-rich foods. Because of the bitter flavour of amino acids, they have rarely been used to reduce GR. We now report the use of a palatable, preformed amino acid mixture in the form of essence of chicken. In all, sixteen healthy male Chinese were served 68 or 136 ml amino acid mixture together with rice, or 15 or 30 min before consumption of white rice. Postprandial blood glucose and plasma insulin concentrations were measured at fasting and every 15 min after consumption of the meal until 60 min after the consumption of the white rice. Subsequent blood samples were taken at 30-min intervals until 210 min. The co-ingestion of 68 ml of amino acid mixture with white rice produced the best results in reducing the peak blood glucose and GR of white rice without increasing the insulinaemic response. It is postulated that amino acid mixtures prime β-cell insulin secretion and peripheral tissue uptake of glucose. The use of ready-to-drink amino acid mixtures may be a useful strategy for lowering the high-GI rice diets consumed in Asia.

scholarly journals Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

1993 ◽  
Vol 177 (3) ◽  
pp. 583-596 ◽  
Author(s):  
P Romagnoli ◽  
C Layet ◽  
J Yewdell ◽  
O Bakke ◽  
R N Germain
Keyword(s):  
Major Histocompatibility Complex ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endocytic Compartments

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

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