Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus.

Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor-mediated endocytosis. MVBs also contained numerous small vesicles, 0.05-0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized triglycerides. Membranes of MVBs contained much more cholesterol and less protein than did Golgi membranes. We conclude that two distinct lipoprotein-filled organelles are located in the bile canalicular pole of hepatocytes. MVBs, a major prelysosomal organelle of low density in the endocytic pathway, contain remnants of triglyceride-rich lipoproteins, whereas secretory vesicles of the Golgi apparatus contain nascent very low density lipoproteins.

Modulation of lipid metabolism at rat hepatic subcellular sites by female sex hormones.

The present studies examine the modulation of lipoprotein metabolism at subcellular sites in the liver by female sex hormones. Subcutaneous injection of ethinyl estradiol (5 mg/kg) decreased both triacylglycerol (TG) lipase activity and neutral cholesteryl ester (CE) hydrolase activity in hepatic endosomes while increasing lysosomal lipid hydrolysis. These data suggest that estrogen may induce a shift in the site of intracellular lipid catabolism similar to that found in fasting animals [1]. This work also shows that TG-lipase activity is increased in the CURL and MVB endosomal fractions of ovariectomized rats compared to that found in the equivalent endosomal compartments of age-matched intact female controls. These observations are consistent with an inhibition of endosomal lipase by female sex hormones under physiologic conditions.

Progressive nuclear translocation of somatostatin analogs.

UNLABELLED: Optimal cancer radiotherapy using Auger electron emitters requires selective localization of radionuclides in close proximity to tumor DNA. METHODS: Intracellular trafficking of (125)I-Tyr1-somatostatin-14 somatotropin-release inhibiting factor (SRIF) and 2 of its analogs, (125)I-WOC 4a and (111)In-pentetreotide, was studied in human neuroblastoma cells. RESULTS: After 24-h incubation, SRIF was degraded or recycled, whereas its protease-resistant analogs progressively accumulated in nuclear fractions. (111)In-pentetreotide binding to DNA increased over time in somatostatin receptor-positive cells but not in somatostatin receptor-negative cells. CONCLUSION: These in vitro studies show that prolonged exposure to radiolabeled SRIF analogs significantly increases their cellular internalization, nuclear translocation, and DNA binding. Clinically, infusion of radiolabeled somatostatin analogs may enhance tumor uptake and retention and provide more effective in situ radiotherapy.

Alterations in lipolytic activity at hepatic subcellular sites of fed and fasted rats.

This study investigates the relationship between the nutritional state of rats and lipid metabolism in distinct hepatic intracellular sites. Hepatic uptake of both protein and triacylglycerol (TG) moieties of injected very low-density lipoprotein (VLDL) is increased in fasted rats compared with fed controls. The VLDL-TG hydrolysis rate is increased in the plasma of fasted rats. This is shown by a higher ratio of labeled free fatty acid (FFA) to TG (FFA/TG). In both fed and fasted rats, a much greater increase of the labeled FFA/TG ratio in endosomes, compared with that in plasma, shows that further TG hydrolysis occurs in prelysosomal compartments. However, in fasted rats, this increase (18-fold) is much less than that in fed rats (69-fold). This observation is supported by the finding of significantly lower TG-lipase activity at pH 5, 7, and 8.6 in the endosomes of fasted rats. In contrast, during fasting, TG-lipase activity in whole liver homogenate and in isolated lysosomes is increased at pH 5. These observations suggest that after feeding there is a shift in intracellular lipolytic activity from lysosomes to prelysosomal organelles.

Use of coca leaf in southern Peru: adaptation or addiction.

Use of coca leaves among the Quechua Indians of the Peruvian altiplano is considered from the biological and social perspectives. Biologically, coca use seems to reduce the loss of body heat in the cold and to enhance working ability to a small degree. It may also have nutritional value. Socially, coca is well integrated into the economic and social systems, providing functions in both spheres. The equation of coca use with cocaine addiction is also considered and a simple equivalence is rejected.

Androgenic modulation of lipid metabolism at subcellular sites in cholestatic rats.

The present studies examine the modulation of lipid metabolism at subcellular sites in the liver of control and cholestatic rats by a male sex hormone. Subcutaneous injection of testosterone enanthate (5 mg/kg body weight), increased triacylglycerol lipase (TG-lipase) and neutral cholesterol ester hydrolase (CE-hydrolase) activity in endosomes of normal rats. During induced cholestasis, TG-lipase and CE-hydrolase activity in endosomal fractions were decreased compared to those in endosomes of age-matched sham-operated female controls. Following testosterone administration to cholestatic rats, endosomal lipolytic enzyme activity increased to approximate the levels of normal controls. These observations may provide insight into the alterations in lipid metabolism which accompany cholestatic liver disease.

Evidence for extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (Fu5AH): a model for delivery of high-density lipoprotein cholesterol.

Rat hepatoma cells (Fu5AH) were studied as a model for the net delivery of apoE-free high-density lipoprotein (HDL) cholesterol to a cell. Incubating cells with HDL results in 1) a decrease in both media-free cholesterol and cholesteryl ester concentration; 2) decreased cell sterol synthesis; and 3) increased cell cholesteryl ester synthesis. HDL cholesteryl ester uptake is increased when cells are incubated for 18 hr in cholesterol poor media. Coincubation of 3H-cholesteryl ester-labeled low-density lipoprotein (LDL) with 50 microM chloroquine or 25 microM monensin results in a decrease in the cellular free cholesterol/cholesteryl ester (FC/CE) isotope ratio, indicating an inhibition in the conversion of cholesteryl ester to free cholesterol. In contrast, chloroquine and monensin do not alter the cellular FC/CE isotope ratio for 3H-CE HDL. This evidence indicates that acidic lysosomal cholesteryl ester hydrolase does not account for the hydrolysis of HDL-CE. Free cholesterol generated from 3H-cholesteryl ester of both LDL and HDL is reesterified intracellularly. At higher HDL concentrations (above 50 micrograms/ml) HDL cholesteryl ester hydrolysis is sensitive to chloroquine. We propose that an extralysosomal pathway is operating in the metabolism of HDL cholesterol and that at higher HDL concentrations a lysosomal pathway may be functioning in addition to an extralysosomal pathway.

Evidence that a neutral cholesteryl ester hydrolase is responsible for the extralysosomal hydrolysis of high-density lipoprotein cholesteryl ester in rat hepatoma cells (Fu5AH).

Diethylumbelliferyl phosphate (UBP) has been shown to inhibit the neutral cholesteryl ester hydrolase activity responsible for hydrolysis of cellular lipid droplet cholesteryl ester (Harrison et al., 1990). The potential for (UBP) to inhibit uptake and hydrolysis of high density lipoprotein (HDL) cholesteryl ester was studied in Fu5AH hepatoma cells, a model for HDL cholesterol delivery. Coincubation of 3H-cholesteryl ester labeled HDL with UBP resulted in a 72% decrease in the cellular free cholesterol/cholesteryl ester (FC/CE) isotope ratio, indicating an inhibition in the conversion of cholesteryl ester to free cholesterol. Total cellular 3H-CE uptake was modestly (27%) but significantly decreased by UBP. Pulse-chase experiments (15 min. pulse and 7 min. chase) were used to study the hydrolysis of HDL 3H-CE in subcellular fractions separated by percoll gradients. The conversion of 3H-CE to 3H-FC could be demonstrated in fractions that comigrated with the plasma membrane/endosome fractions but were well separated from lysosomes. Neutral cholesteryl ester hydrolase activity was detected in those same fractions. These results suggest that an extralysosomal pathway is operating in the metabolism of HDL cholesterol and its delivery to hepatoma cells.

A role for retrosomes in intracellular cholesterol transport from endosomes to the plasma membrane.

The recycling component (retrosome) of the endocytic pathway was evaluated as a potential vehicle for the recycling of lipoprotein-derived cholesterol and the maintenance of a high concentration of free cholesterol in plasma membranes. Receptor-to-ligand ratios were established in three distinct endosomal compartments using a recycling receptor (apolipoprotein B/E) to confirm isolated retrosomes as recycling vesicles. Compositional studies showed that retrosomes have twice the free cholesterol in their limiting membranes as do the endosomal compartments from which they derive. Furthermore, of the three isolated endosomal fractions, retrosomes showed the highest ratio of free to esterified cholesterol derived from injected very low density lipoprotein as well as the highest free-to-esterified cholesterol mass ratio overall, confirming endosomal cholesteryl ester hydrolysis and sorting. Endosomal neutral cholesterol esterase was identified by immunoblot, whereas electron microscopy employing membrane cholesterol-specific filipin revealed a high concentration of cholesterol in appendages that appear to be the formative stage of retrosomal biogenesis.

Effect of carpaine, a papaya alkaloid, on the circulatory function in the rat.

Carpaine is one of the major components of alkaloid of papaya leaves. Circulatory effects of carpaine were studied in Wistar male rats weighing 314 +/- 13 g, under pentabarbital (30 mg/kg) anesthesia. Increasing dosages of carpaine from 0.5 mg/kg to 2.0 mg/kg resulted in progressive decrease in systolic, diastolic, and mean arterial blood pressure. Selective autonomic nervous blockade with atropine sulfate (1 mg/kg) or propranolol hydrochloride (8 mg/kg) did not alter the circulatory response to carpaine. Carpaine, 2 mg/kg, reduced cardiac output, stroke volume, stroke work, and cardiac power, but the calculated total peripheral resistance remained unchanged. It is concluded from these results that carpaine affects the myocardium directly. The effects of carpaine may be related to its macrocyclic dilactone structure, a possible cation chelating structure.

Triacylglycerol hydrolysis in isolated hepatic endosomes.

Three endosomal compartments including the compartment for uncoupling receptor and ligand (CURL), multivesicular bodies (MVB), and a putative recycling fraction (retrosomes) were isolated from rat liver homogenates fifteen minutes after a bolus injection of very low density lipoprotein (VLDL) was delivered into a femoral vein. Assays for enzyme markers indicate a minimal contamination with either lysosomes or Golgi. The increase in specific activity of the radiolabeled ligand (VLDL) during the isolation procedure from homogenate to MVB, demonstrates a 200-250-fold purification of this organelle. All three fractions have the ability to catabolize triacylglycerol substrate both as triolein and as VLDL triacylglycerol. Furthermore, incubation of isolated endosomes following injection of endogenously labeled VLDL demonstrate their ability to hydrolyze VLDL triacylglycerol in situ. Three distinct lipolytic pH optima were found at pH 5.5, 7.1, and 8.6. The effects of serum, MgCl2, CaCl2, NaCl, sodium dodecyl sulfate, bile acids, and antibody to hepatic triacylglycerol lipase on the individual endosome fractions demonstrated distinct lipolytic activities in the different compartments. Results indicate that both an endosomal neutral lipase as well as hepatic triacylglycerol lipase make a significant contribution to lipolytic processing of endocytosed lipoproteins prior to their resecretion of further processing in hepatic lysosomes.

Secretion of lipoproteins from the liver of normal and Watanabe heritable hyperlipidemic rabbits.

We compared the rate of accumulation of lipoproteins in perfusates of isolated livers from normal New Zealand White rabbits and Watanabe heritable hyperlipidemic (WHHL) rabbits, in which a gene mutation has produced a virtually complete deficiency of low density lipoprotein (LDL) receptors. The rate of accumulation of apolipoprotein B-100 did not differ in perfusates of livers from normal and mutant animals and little or no apolipoprotein B-48 was detected. In both groups, virtually all apolipoprotein B accumulated in very low density lipoprotein (VLDL). Experiments in which [3H]lysine was added to the perfusates showed that the apolipoprotein B that accumulated in VLDL was newly synthesized by the liver whereas the small amount of apolipoprotein B found in lipoproteins of higher density appeared to be washed out of extravascular spaces during perfusion. Perfusate VLDL from both groups contained more triglycerides and less cholesteryl esters than their counterparts from blood plasma. As compared with perfusate VLDL from normal livers, those from livers of WHHL rabbits were enriched in cholesteryl esters. Experiments in which Triton WR-1339 was injected into the blood of intact rabbits confirmed the observations with perfused livers. Previous studies have shown that the extent to which VLDL is converted to LDL is increased several-fold in WHHL rabbits. Taken together with our present results, which fail to provide evidence for increased secretion of apolipoprotein B or de novo secretion of lipoproteins other than VLDL that contain apolipoprotein B, it can be concluded that overproduction of LDL in rabbits lacking LDL receptors is solely the result of altered metabolism of VLDL.

Effect of chloroquine on low-density lipoprotein catabolic pathway in rat hepatocytes.

Injection of 125I-low-density lipoprotein (LDL) into estradiol-treated rats substantially increases the volume of the multivesicular body compartment in hepatocytes. When these animals were additionally given chloroquine 60 and 120 min before injection of 125I-LDL, the amount of 125I retained by the liver increased threefold and the amount of label in multivesicular bodies increased fivefold, as determined by quantitative analysis of autoradiograms. Lamellar inclusions that appeared after chloroquine also contained a substantial fraction of 125I, whereas the fraction of 125I in secondary lysosomes was reduced. From these data it is concluded that the multivesicular bodies comprise an intermediate endocytic compartment, the conversion of which to secondary lysosomes is impeded by chloroquine.

Metabolism of apoE-free high density lipoproteins in rat hepatoma cells: evidence for a retroendocytic pathway.

The cellular metabolism of apoE-free HDL (HDL) was studied in rat hepatoma cells (FU5AH). Cells incubated with HDL showed a dose-dependent decreased incorporation of [14C]acetate into cell sterol, indicating a net cholesterol delivery to the cells. HDL was localized both at the cell surface and inside the cell. This conclusion was drawn from both the association of 125I-labeled HDL with the cells under different experimental conditions and morphological evidence based on the association of colloidal gold-labeled HDL with the cells. Up to 63% of the 125I-labeled HDL protein initially inside the cell was subsequently recovered in the media as trichloroacetic acid precipitable (TCA-ppt) protein after a 30-min, 37 degrees C chase with a 100-fold concentration of unlabeled HDL. About 27% of the TCA-ppt apoprotein originally inside the cell was recovered as TCA-soluble material. Thus, we conclude that of the HDL apoprotein taken up by the cells, the majority is resecreted by a retroendocytosis pathway. The quantity of HDL apoprotein reappearing in the media was stimulated by the presence of unlabeled HDL in the media, while the amount of TCA-soluble material produced was not. Retroendocytosis of HDL was inhibited at 0 degree C and by the presence of 10 mM NaCN, 20 mM 2-deoxy-D-glucose in the media. Thus, the pathway appears to be both temperature- and energy-sensitive. HDL resecreted by the cell were depleted of cholesteryl ester and showed an altered size distribution, indicative of lipoprotein catabolism and remodeling. This study provides evidence for the existence of an endocytosis-retroendocytosis pathway for HDL apoproteins in a rat hepatoma cell and for the possibility that the endocytosis-retroendocytosis pathway may be involved in lipid delivery to the cell.

Multivesicular bodies isolated from rat hepatocytes. Cytochemical evidence for transformation into secondary lysosomes by fusion with primary lysosomes.

Plasma lipoproteins (and other ligands) are endocytosed by hepatocytes and appear in multivesicular bodies (MVBs) in the Golgi-lysosome region of the cell prior to their degradation. We have isolated MVB fractions from livers of estradiol-treated rats, permitting studies of their properties (Hornick et al. 1985). Here we report our cytochemical studies of lysosomal enzyme activity in partially and highly purified MVB fractions and in MVBs in hepatocytes in situ. Only about 15% of partially or highly purified MVBs were positive for acid phosphatase and arylsulfatase, consistent with the prelysosomal nature of this compartment. Partially purified MVB fractions contained small round vesicles, 70-120 nm in diameter, which stained intensely for these enzymes; occasionally these vesicles appeared to fuse with MVBs, suggesting that these structures are primary lysosomes. Such stained vesicles were rarely seen in highly purified MVB preparations. Acid phosphatase reaction product with cerium as capture reagent appeared as uniform precipitates surrounding endocytosed plasma lipoproteins in positively stained MVBs. Arylsulfatase reaction product, however, appeared as distinctive arc or plaque-like deposits just inside the MVB-limiting membrane, often in continuity with intense reaction product contained in a fusing primary lysosome. Similar putative primary lysosomes were occasionally observed in isolated, "intact" Golgi fractions from the same livers. Similar histochemical reactivities of MVBs and putative primary lysosomes were observed in thin sections of hepatocytes in situ. These observations support the conclusion that, in hepatocytes, MVBs represent the immediate prelysosomal compartment in the endocytic pathway of macromolecular catabolism, and suggest that MVBs are converted to secondary lysosomes by direct fusion with primary lysosomes arising from closely adjacent Golgi compartments.

Lipoproteins and apolipoproteins in postmortem serum.

In order to correlate lipoprotein and apolipoprotein profiles with atherosclerotic lesions at autopsy, the feasibility of using postmortem serum in these determinations was evaluated. Lipoprotein and apolipoprotein profiles in dogs and monkeys were compared in pre- and postmortem serum samples, and it was found that, up to 24 h postmortem, no appreciable differences were observed in apolipoprotein A-I, B, E, and A-IV serum concentration when compared to premortem values. Electrophoretic mobilities and apolipoprotein gradient gel immunoblotting also revealed no differences in apolipoprotein size distribution. We conclude that postmortem sampling up to 24 h can be used effectively to approximate premortem lipoprotein and apolipoprotein profiles provided that careful sampling techniques are observed.

Hepatic uptake of chylomicron remnants in WHHL rabbits: a mechanism genetically distinct from the low density lipoprotein receptor.

Homozygous Watanabe hereditary hyperlipidemic (WHHL) rabbits have a near-complete deficiency of low density lipoprotein (LDL) receptors in liver and other tissues. As a result, these rabbits clear LDL from plasma at an abnormally slow rate. In the current studies we show that WHHL rabbits clear chylomicrons from plasma at a normal rate. Chylomicrons are cleared by a two-step process: (i) hydrolysis of triglycerides in extrahepatic tissues to yield cholesteryl ester-rich remnant particles and (ii) rapid uptake of the remnants by liver. Normal and WHHL rabbits were given intravenous injections of rat chylomicrons labeled either in the lipid portion with [3H]cholesterol and [14C]palmitate or in the protein portion with [125]iodine. All radiolabeled components were removed from plasma at comparable rates in normal and WHHL rabbits. Comparable amounts of radioactivity accumulated in livers of animals from both genotypes. In vitro assays showed that liver membranes from WHHL rabbits were markedly deficient in the binding of 125I-labeled chylomicron remnants as well as 125I-labeled LDL, implying that chylomicron remnants can bind to the hepatic LDL receptor. We conclude that the rabbit liver normally has at least two genetically distinct lipoprotein uptake mechanisms, both of which recognize chylomicron remnants: (i) the LDL receptor and (ii) a specific chylomicron remnant uptake mechanism that is not measured adequately by current in vitro membrane binding assays. WHHL rabbits possess a normal chylomicron remnant uptake mechanism that allows them to clear chylomicrons from plasma at a rapid rate despite their genetic deficiency of LDL receptors.

Hepatic metabolism of oxidatively modified apo E-free high-density lipoproteins.

AIMS/BACKGROUND: The metabolism of rat apo E-free high-density lipoproteins (HDL) was contrasted with oxidatively modified apo E-free high-density lipoproteins (OX-HDL) in the rat hepatoma cell, Fu5AH. RESULTS: When 10-100 microg/ml [125I]-HDL or [125I]-OX-HDL were incubated with cells for 4 h at 37 degrees C, cellular uptake of oxidized lipoproteins was twice control. In contrast, protein degradation was equal. [125I]-HDL or [125I]-OX-HDL were incubated with the cells for 4 h followed by a 4 h chase with unlabeled HDL and OX-HDL, respectively. In these experiments, 80% of [125I]-HDL was resecreted from the cell within 30 min while 50% of [125I]-OX-HDL was retained by the cell after 2 h. Electron microscopy was used to determine if the OX-HDL was retained in lysosomes. Cells were incubated with gold-labeled OX-HDL, and lysosomes were stained with acid phosphatase. Gold-labeled OX-HDL was abundant in intracellular vesicles that were not reactive to acid phosphatase. However, vesicles with a high content of OX-HDL frequently stained positively for 3,3'-diaminobenzidine, a stain that reacts with catalase and is used to detect peroxisomes. CONCLUSIONS: The present evidence indicates that the cellular metabolism of OX-HDL is different from that of unmodified HDL.

Identification and characterization of ATP-dependent proton transport by rat liver multivesicular bodies.

Multivesicular bodies (MVB), prelysosomal organelles in the endocytic pathway, were prepared from estrogen-treated rat livers and examined for the presence of ATP-dependent proton transport. Vesicle acidification, assessed by acridine orange fluorescence quenching, was ATP dependent (ATP much greater than GTP, UTP), was enriched 25-fold over homogenate, was abolished by pretreatment with protonophores or a nonionic detergent, exhibited a pH optimum of 7.5, was inhibited by N-ethylmaleimide (NEM) (IC50 approximately 5 microM) and N,N'-dicyclohexylcarbodiimide (IC50 approximately 5 microM), and was resistant to inhibition by vanadate, ouabain, and oligomycin. Acidification exhibited no specific cation requirement; however, maximal rates of acidification depended upon the presence of Cl- (Km approximately 20 mM). Other anions were less effective in supporting acidification (Cl- greater than Br- greater than much greater than gluconate, NO-3, SO2-4, and mannitol), and indeed NO-3 inhibited acidification even in the presence of 150 mM Cl-. The proton transport mechanism appeared to be electrogenic based on: (a) enhancement of acidification by valinomycin in the presence of K gluconate, and (b) ATP-dependent fluorescence quenching of bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol, a membrane potential-sensitive anionic dye. Furthermore, the magnitude of the pH and electrical gradients generated by the proton transport mechanism appeared to vary inversely in the presence and absence of Cl-. Finally, MVB exhibited ATPase activity that was resistant to ouabain and oligomycin, but was inhibited 32.3% by 1 mM NEM, 33.7% by 200 microM dicyclohexylcarbodiimide, and 18.7% by KNO3. In isolated MVB, therefore, the NEM-sensitive ATPase activity may represent the enzymatic equivalent of a proton pump. These studies identify and characterize an ATP-dependent electrogenic proton transport process in rat liver MVB which shares many of the properties of the proton pump described in clathrin-coated vesicles, endosomes, lysosomes, Golgi, and endoplasmic reticulum from liver and other tissues. Acidification of MVB differed somewhat from that of rat liver clathrin-coated vesicles in response to Br- and NO-3, suggesting that membrane properties of these two organelles might differ.