Golgi-endosomal Fractions Research Articles

Processing of the insulin-like growth factor-II–mannose 6-phosphate receptor in isolated liver subcellular fractions

A truncated, soluble form of the insulin-like growth factor-II-mannose 6-phosphate (IGF-II-M6P) receptor has been identified in serum and shown to be released from cultured tissues and cells, liver being the main contributor to serum receptor in adult rats. In the present study, the processing of the IGF-II-M6P receptor has been characterized in isolated liver subcellular fractions using ligand binding, affinity crosslinking, and Western immunoblotting techniques. The receptor in plasma membrane fractions differed from that in Golgi-endosomal fractions by: (i) a lower molecular size upon reducing polyacrylamide gel electrophoresis (245 vs. 255 kDa); (ii) a less tight membrane association as judged upon extractibility by NaCI; and (iii) the inability to recognize antibody anti-22C, directed against the cytoplasmic domain of the receptor. Incubation of cell fractions at 30 degrees C led to a pH- and time-dependent release of the receptor into the medium. The pH optimum for release was 5.5 in the Golgi-endosomal fraction and 7.5 in plasma membrane fractions; at this pH, approximately 2% and 20%-30% of total receptors were released per hour, respectively. Receptor release was inhibited in a dose-dependent manner by aprotinin, benzamidine, and leupeptin in the Golgi-endosomal fraction, and by 1,10 phenanthroline in plasma membrane fractions, although high concentrations were required for inhibition. The receptor released from Golgi-endosomes showed a 5-10 kDa reduction in size and a loss of ability to recognize antibody anti-22C, but that released from plasma membranes showed little or no changes in size. We conclude that soluble, carboxy-terminally truncated forms of the IGF-II-M6P receptor are generated from the intact receptor in isolated Golgi-endosomal and plasma membrane fractions. However, receptor processing in these fractions exhibits different properties, suggesting the involvement of different proteases.

Characterization of an in Vivo Hormonally Regulated Phosphodiesterase 3 (PDE3) Associated with a Liver Golgi-Endosomal Fraction

The biochemical properties of an in vivo hormonally regulated low Km cAMP phosphodiesterase (PDE) activity associated with a liver Golgi-endosomal (GE) fraction have been characterized. DEAE-Sephacel chromatography of a GE fraction solubilized by a lysosomal extract resulted in the sequential elution of three peaks of activity (numbered I, II, and III), while ion-exchange HPLC resolved five peaks of activity (numbered 1, 2, 3, 4, and 5). Based on the sensitivity of the eluted activity to cGMP and selected phosphodiesterase inhibitors, two phosphodiesterase isoforms were resolved: a cGMP-stimulated and EHNA-inhibited PDE2, eluted in DEAE-Sephacel peak I and HPLC peak 2 and a cGMP-, a cilostamide-, and ICI 118233-inhibited PDE3, eluted in DEAE-Sephacel peak III and HPLC peaks 3, 4, and 5. GE fractions isolated after acute treatments with insulin, tetraiodoglucagon, and growth hormone displayed an increase in phosphodiesterase activity relative to saline-injected controls, as did GE fractions from genetically obese and hyperinsulinemic rats relative to lean littermates. In all experimental rats, an increase in PDE3 activity associated with DEAE-Sephacel peak III and HPLC peaks 4 and 5 was observed relative to control animals. Furthermore, in genetically obese Zucker rats, an increase in the sensitivity of PDE activity to cilostamide and in the amount of PDE activity immunoprecipitated by an antibody to adipose tissue PDE3 was observed relative to lean littermates. These results extend earlier studies on isolated hepatocytes and show that liver PDE3 is the main if not sole PDE isoform activated by insulin, glucagon, and growth hormone in vivo.

Insulin-induced redistribution of the insulin-like growth factor II/mannose 6-phosphate receptor in intact rat liver.

The ability of acute insulin treatment to elicit a redistribution of the liver insulin-like growth factor-II/ mannose 6-phosphate (IGF-II/M6P) receptor has been studied in rats, using cell fractionation. Injection of insulin (0.4-50 microg) led to a time- and dose-dependent decrease in IGF-II binding activity in Golgi-endosomal (GE) fractions, along with an increase in activity in the plasma membrane (PM) fraction; only receptor number was affected. Quantitative subfractionation of the microsomal fraction on sucrose density gradients showed that IGF-II binding activity distributed similarly to galactosyltransferase (a Golgi marker), at slightly higher densities than in vivo internalized (125)I-insulin, and at lower densities than 5' nucleotidase and alkaline phosphodiesterase (two plasma membrane markers). Insulin treatment led to a slight time-dependent and reversible shift of IGF-II binding activity toward higher densities. Subfractionation of the GE fraction on Percoll gradients showed that IGF-II binding activity was broadly distributed, with about 60% at low densities coinciding with galactosyltransferase and early internalized (125)I-insulin and with 40% at high densities in the region of late internalized (125)I-insulin. Insulin treatment caused a time-dependent and reversible shift of the distribution of IGF-II binding activity toward low densities. On SDS-PAGE, the size of the affinity-labeled IGF-II/M6P receptor was comparable in GE and PM fractions (about 255 kDa), but on Western blots receptor size was slightly lower in the latter (245 kDa) than in the former (255 kDa). Insulin treatment did not affect the size, but modified the abundance of the IGF-II/M6P receptor in a manner similar to that of IGF-II binding. In vivo chloroquine treatment fully suppressed the changes in IGF-II binding activity in liver GE and PM fractions observed in insulin-treated rats. We conclude that insulin elicits a time-dependent and reversible redistribution of liver IGF-II receptors from Golgi elements and endosomes to the plasma membrane, presumably via early endosomes.

Activation of a cGMP‐stimulated cAMP phosphodiesterase by protein kinase C in a liver Golgi–endosomal fraction

The ability of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) to stimulate cAMP phosphodiesterase (PDE) activity in a liver Golgi-endosomal (GE) fraction was examined in vivo and in a cell-free system. Injection into rats of 4 beta-phorbol 12-myristate 13-acetate, a known activator of PKC, caused a rapid and marked increase in PKC activity (+325% at 10 min) in the GE fraction, along with an increase in the abundance of the PKC alpha-isoform as seen on Western immunoblots. Concurrently, 4 beta-phorbol 12-myristate 13-acetate treatment caused a time-dependent increase in cAMP PDE activity in the GE fraction (96% at 30 min). Addition of the catalytic subunit of protein kinase A (PKA) to GE fractions from control and 4 beta-phorbol 12-myristate 13-acetate-treated rats led to a comparable increase (130-150%) in PDE activity, suggesting that PKA is probably not involved in the in-vivo effect of 4 beta-phorbol 12-myristate 13-acetate. In contrast, addition of purified PKC increased (twofold) PDE activity in GE fractions from control rats but affected only slightly the activity in GE fractions from 4 beta-phorbol 12-myristate 13-acetate-treated rats. About 50% of the Triton-X-100-solubilized cAMP PDE activity in the GE fraction was immunoprecipitated with an anti-PDE3 antibody. On DEAE-Sephacel chromatography, three peaks of PDE were sequentially eluted: one early peak, which was stimulated by cGMP and inhibited by erythro-9 (2-hydroxy-3-nonyl) adenine (EHNA); a selective inhibitor of type 2 PDEs; and two retarded peaks of activity, which were potently inhibited by cGMP and cilostamide, an inhibitor of type 3 PDEs. Further characterization of peak I by HPLC resolved a major peak which was activated (threefold) by 5 microM cGMP and inhibited (87%) by 25 microM EHNA, and a minor peak which was insensitive to EHNA and cilostamide. 4 beta-Phorbol 12-myristate 13-acetate treatment caused a selective increase (2.5-fold) in the activity associated with DEAE-Sephacel peak I, without changing the K(m) value. These results suggest that PKC selectively activates a PDE2, cGMP-stimulated isoform in the GE fraction.

Ligand-mediated internalization of glucagon receptors in intact rat liver.

The ligand-induced internalization of the hepatic glucagon receptor has been studied in rats in vivo using cell fractionation. Injection of glucagon (11 nmol/100 g BW) led to a 2- to 3-fold increase in glucagon-binding activity in Golgi-endosomal (GE) fractions along with a 10-20% decrease in binding activity in plasma membrane (PM) fractions. These changes were time and dose dependent, reaching a maximum by 12-24 min and undergoing reversal in 2 h. Glucagon injection also caused a 20% decrease in glucagon binding to the total particulate fraction, which did not occur when binding was measured in the presence of the detergent octylglucoside. The change in glucagon-binding activity in PM and GE fractions resulted mainly from a change in receptor number; affinity remained unaffected (apparent Kd, 0.5 and 5 nM, respectively). A 5- to 10-fold increase in the glucagon content of GE fractions was observed in glucagon-treated rats. Neither the distribution of PM and Golgi marker enzymes nor that of the asialoglycoprotein receptor was affected by glucagon treatment. Regardless of glucagon treatment, glucagon receptors in GE fractions were less sensitive to GTP than receptors in PM fractions with respect to both inhibition of steady state binding and dissociation of prebound ligand. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, glucagon-receptor complexes formed in PM and GE fractions and subsequently cross-linked showed the same apparent mol wt (57 kilodaltons). In addition, they were identically sensitive to N-glycanase treatment, with two major species of lower mol wt generated. However, only cross-linked complexes associated with PM fractions showed detectable GTP sensitivity. GE fractions displayed a GTP-sensitive adenylate cyclase activity that was about 12 times lower than that of PM fractions. In both fractions, activity was stimulated by the addition of forskolin (8-fold) and, to a lesser extent, glucagon (3-fold). In vivo glucagon treatment led to an increase in activity in GE, but not PM, fractions. These results are consistent with the view that upon acute occupancy, hepatic glucagon receptors are rapidly and specifically internalized along with their ligand. During this process, receptor retained structural integrity and uncouple, albeit partially, from other components of the adenylate cyclase system.

Mechanisms of up-regulation of the liver insulin receptor in chronically hypoinsulinemic rats: assessment of receptor endocytosis

Chronic hypoinsulinemic states in rodents are known to cause an increase in the number of insulin receptors at the hepatocyte surface. To assess whether this change results from a reduced endocytosis of the receptors, the effects of streptozotocin treatment and fasting on the number and the subcellular distribution of hepatic insulin receptors have been evaluated in the rat. In streptozotocin-treated rats, insulin receptor number was increased by 25–40% in plasma membrane and total cellular membrane fractions, and by 60–130% in the light Golgi-endosomal (GE) fraction. In contrast, receptor number was unaffected in the intermediate GE fraction and decreased by 25–35% in the heavy GE fraction. Such changes were detectable at 12 h in GE fractions and at 2 days in other subcellular fractions, and lasted for at least 8 days. Streptozotocin treatment also led to a 3- to 4-fold decrease in the insulin content of GE fractions, indicating reduced hormone endocytosis. Fasting for 16 h elicited changes in receptor and ligand concentration in cell fractions comparable to those induced by Streptozotocin. It is concluded that, although endocytosis of hepatic insulin receptors is reduced in chronic hypoinsulinemic states, changes in receptor synthesis and/or degradation also occur in these states.

Degradation of glucagon in isolated liver endosomes. ATP-dependence and partial characterization of degradation products

Endosomes have recently been identified as one major site of glucagon degradation in intact rat liver. In this study, a cell-free system has been used to assess the role of ATP-dependent acidification in endosomal glucagon degradation and identify the glucagon products generated. Percoll gradient fractionation of Golgi-endosomal fractions prepared 10-30 min after injection of [125I]iodoglucagon showed a time-dependent shift of the radioactivity towards high densities. Regardless of time, the radioactivity was less precipitable by trichloroacetic acid (Cl3Ac) at high densities than at low densities. Chloroquine treatment slightly increased the density shift of the radioactivity and decreased its Cl3Ac-precipitability throughout the gradient. Incubation of endosomal fractions containing [125I]iodoglucagon in 0.15 M-KCl at 30 degrees C resulted in a time- and pH-dependent generation of Cl3Ac-soluble radioactivity, with a maximum at pH 4 (t1/2, 7 min). At pH 5, 1,10-phenanthroline, bacitracin and p-chloromercuribenzoic acid partially inhibited [125I]iodoglucagon degradation. At pH 6-7, ATP stimulated [125I]iodoglucagon degradation by 5-10-fold and caused endosomal acidification as judged from Acridine Orange uptake. The effects of ATP were inhibited by chloroquine, monensin, N-ethylmaleimide and dansylcadaverine. Poly(ethylene glycol) (PEG) precipitation of the radioactivity associated with endosomes showed that lowering the pH below 5.5 caused dissociation of the glucagon-receptor complex, and that, regardless of incubation conditions, all degraded [125I]iodoglucagon diffused extraluminally. On h.p.l.c., at least three products less hydrophobic than [125I]iodoglucagon were identified in incubation mixtures along with monoiodotyrosine. Radiosequence analysis of the products revealed one major cleavage located C-terminally to Tyr-13 and two minor cleavages affecting Thr-5-Phe-6 and Phe-6-Thr-7 bonds. It is concluded that glucagon degradation in liver endosomes is functionally linked to ATP-dependent endosomal acidification and involves several cleavages in the glucagon sequence.

Activation of rat liver adenylate cyclase by cholera toxin requires toxin internalization and processing in endosomes

Involvement of acidic cell compartments in processing and action of cholera toxin (CT) in rat liver has been examined using subcellular fractionation. Liver cell fractions prepared various times after CT injection display, after a lag phase, a progressive increase in adenylate cyclase activity, detectable earlier in Golgi-endosomal fractions (20 min) than in plasma membrane fractions (30 min), with a maximum (3-fold basal activity) achieved by 60-90 min. Endosomes containing in vivo internalized CT display a time-dependent increase in their ability to bind anti-A-subunit antibodies and to stimulate exogenous adenylate cyclase, which kinetically parallels the generation of A1 peptide, suggesting a translocation of A-subunit (or A1 peptide) across the endosomal membrane. In vivo chloroquine treatment inhibits endocytosis of CT taken up into the liver, lengthens the lag phase for adenylate cyclase activation by CT, and reduces by 3- to 10-fold the apparent affinity of the toxin for the enzyme. Incubation of endosomes containing internalized toxin at 37 degrees C under isotonic conditions results in a pH-dependent increase in generation of A1 peptide, membrane translocation of A-subunit (or A1 peptide), and degradation of toxin, with a maximum at pH 5. Addition of ATP, by decreasing the internal endosomal pH, stimulates both generation of the A1 peptide and degradation of toxin at pH 6-8. It is concluded that activation of adenylate cyclase by CT in intact liver requires association and subsequent processing of toxin in an acidic cell compartment, presumably endosomal.

Characterization of a Liver Low Michaelis-Menten Constant 3′,5′-Cyclic Adenosine Monophosphate Phosphodiesterase Activity Sensitive to Thyroid Status*

These studies were undertaken to assess the subcellular distribution and some biochemical properties of the hepatic cAMP phosphodiesterase(s) whose activity is modulated by the thyroid status in the rat. Thyroidectomy led to a 2-fold increase in low Michaelis-Menten constant (Km) cAMP phosphodiesterase activity in Golgi-endosomal fractions, but little affected this activity in crude particulate fractions. On analytical sucrose density gradients, an increase in cAMP phosphodiesterase activity in particulate elements which equilibrated at densities 1.17-1.22 was also observed. Acute insulin treatment did not further increase cAMP phosphodiesterase activity in Golgi-endosomal fractions of thyroidectomized rats. Up to 75% of the cAMP phosphodiesterase activity associated with Golgi-endosomal fraction of euthyroid and hypothyroid rats was inhibited by cGMP (IC50, 10 microM and 1 microM, respectively). Activity was also potently inhibited by griseolic acid, cilostamide, and cilostazole (IC50, less than 1 microM) but was much less sensitive to R0-20-1724 (IC50, 1 mM). Treatment of Golgi-endosomal fractions by a hypotonic extract of rat liver lysosomes led to the solubilization of 50% of low Km cAMP phosphodiesterase activity. On sucrose density gradients, the solubilized activity migrated as a slightly asymmetrical peak with a sedimentation coefficient of 6 S in euthyroid rats and 6.9 S in hypothyroid rats. On nondenaturing polyacrylamide gel electrophoresis, the activity migrated as two majors peaks with Rf values of 0.23 and 0.50; only the activity associated with the fast-moving peak was increased by thyroidectomy. On diethylaminoethyl-Sephacel chromatography, four peaks of cAMP phosphodiesterase activity, two of which were cGMP-inhibitable, were resolved. Thyroidectomy increased the activity associated with one of the cGMP-inhibitable peaks (eluted at 0.7-0.9 M sodium acetate) and led to the appearance of a new peak of activity (eluted at 0.4 M), which was not sensitive to cGMP. These results show that the low Km phosphodiesterase activity associated with liver Golgi-endosomal fractions, previously shown to be increased in hyperinsulinemic rats, is also increased in hypothyroid animals. They also suggest that, based on pharmacological and physical criteria, the enzyme species affected by the thyroid status belongs to the cGMP-and cilostamide-inhibited subclass of low Km cAMP phosphodiesterases.

Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment

The uptake and processing of glucagon into liver endosomes were studied in vivo by subcellular fractionation. After injection of [[125I]iodo-Tyr10]glucagon and [[125I]iodo-Tyr13]glucagon to rats, the uptake of radioactivity into the liver was maximum at 2 min (6% of the dose/g of tissue). On differential centrifugation, the radioactivity in the homogenate was recovered mainly in the nuclear (N), microsomal (P) and supernatant (S) fractions, with maxima at 5, 10 and 40 min, respectively; recovery of radioactivity in the mitochondrial-lysosomal (ML) fraction did not exceed 6% and was maximal at 20 min. On density-gradient centrifugation, the radioactivity associated first (2-10 min) with plasma membranes and then (10-40 min) with Golgi-endosomal (GE) fractions, with 2-5-fold and 20-150-fold enrichments respectively. Subfractionation of the GE fractions showed that, unlike the Golgi marker galactosyltransferase, the radioactivity was density-shifted by diaminobenzidine cytochemistry. Subfractionation of the ML fraction isolated at 40 min showed that more than half of the radioactivity was recovered at lower densities than the lysosomal marker acid phosphatase. Throughout the time of study, the [125I]iodoglucagon associated with the P, PM and GE fractions remained at least 80-90% trichloroacetic acid (TCA)-precipitable, whereas that associated with other fractions, especially the S fraction, became progressively TCA-soluble. On gel filtration and h.p.l.c., the small amount of degraded [125I]iodoglucagon associated with GE fractions was found to consist of monoiodotyrosine. Chloroquine treatment of [125I]iodoglucagon-injected rats caused a moderate but significant increase in the late recovery of radioactivity in the ML, P and GE fractions, but had little effect on the association of the ML radioactivity with acid-phosphatase-containing structures. Chloroquine treatment also led to a paradoxical decrease in the TCA-precipitability of the radioactivity associated with the P and GE fractions. Upon h.p.l.c. analysis of GE extracts of chloroquine-treated rats, at least four degradation products less hydrophobic than intact [125I]iodoglucagon were identified. Radio-sequence analysis of four of these products revealed three cleavages, affecting bonds Ser2-Gln3, Thr5-Phe6 and Phe6-Thr7. When GE fractions containing internalized [125I]iodoglucagon were incubated in iso-osmotic KCl at 30 degrees C, a rapid generation of TCA-soluble products was observed, with a maximum at pH 4. We conclude that endosomes are a major site at which internalized glucagon is degraded, endosomal acidification being required for optimum degradation.

Effects of a long-lasting hyperinsulinemia induced by insulin infusion on the subcellular distribution of liver insulin receptors in the rat.

Acute hyperinsulinemia in rats have been shown to cause enhanced endocytosis of liver insulin receptors with little or no change in the total receptor number. To determine whether a similar phenomenon occurs in long-lasting hyperinsulinemia, the subcellular distribution of liver insulin receptors has been studied in rats infused continuously with insulin (0.4 and 0.2 U/h) for 4 days. In rats in which plasma insulin concentration was maintained at 15-20 ng/ml, there was, from 3 to 24 h, a 2-fold decrease in insulin binding to plasma membranes (PM), along with 2 to 4-fold increase in insulin binding to the light (GEI), intermediate (GEi) and heavy (GEh) Golgi-endosomal fractions; concomitantly, there was a 10-fold increase in the insulin content of Golgi-endosomal fractions. After 24 h, the changes in insulin binding to PM and GEI were maintained, but the increase in both insulin binding activity and insulin content of GEi and GEh became progressively less marked, although plasma insulin concentration remained elevated. Throughout infusion, insulin binding to the total particulate fraction was unchanged. In rats, in which plasma insulin was maintained at 6-8 ng/ml, insulin binding to PM was decreased to a lesser degree and insulin binding to Golgi-endosomal fractions was unchanged (GEh) or decreased (GEI and GEi), although the insulin content of these fractions remained high. These results suggest that, while an enhanced receptor endocytosis accounts for the decrease in cell surface receptors observed at an early stage of the hyperinsulinemia, additional regulatory mechanisms are probably involved at a later stage.

Insulin-dependent changes in subcellular distribution of liver insulin receptors in obese Zucker rats.

Acute hyperinsulinaemias induced by insulin and stimulants of insulin secretion have been shown to cause a translocation of liver insulin receptors from the cell surface to the intracellular compartment, with little or no change in total receptor number. To determine whether a similar phenomenon occurs in chronic hyperinsulinaemic states, we have carried out a longitudinal study of total, cell surface and intracellular liver insulin receptors in genetically obese Zucker rats, with spontaneously develop hyperinsulinaemia. Liver plasma membranes, Golgi-endosomal fractions, a microsomal fraction and a total particulate fraction were isolated in 2-14-week old obese (fa/fa) rats and examined for specific insulin binding relative to lean (Fa/?) age-matched animals. In 16-day old rats, which were still normoinsulinaemic, insulin binding was unchanged. Later on, as hyperinsulinaemia developed, three sequential changes in insulin binding activity were observed: first, a 25-30% increase in Golgi-endosomal fractions (20 days); then, a 50-60% decrease in Golgi-endosomal fractions (4-5 weeks); and finally, a 50% decrease in plasma membranes (11 weeks), microsomal fraction and total particulate fraction (14 weeks), accompanied by restoration in Golgi-endosomal fractions (8-11 weeks). Unlike insulin receptors, insulin extractable from Golgi-endosomal fractions at 4-5 weeks was unchanged or increased. We conclude that, although an early increase in the endocytosis of liver insulin receptors may occur in hyperinsulinaemic Zucker rats, this mechanism does not account for the later decrease in cell surface receptors observed in these animals.

Characterization of rat liver endosomal fractions. In vivo activation of insulin-stimulable receptor kinase in these structures.

A protocol employing discontinuous sucrose gradient centrifugation was developed to prepare light mitochondrial (L) and Golgi fraction endosomes from simultaneously prepared parent L and microsomal fractions. As judged by the concentration of labeled hormone postinjection, L intermediate and heavy endosome subfractions were 40- to 175-fold purified and Golgi intermediate and heavy endosome subfractions were 30- to 45-fold purified. On electron microscopy, L endosomal fractions contained a predominance of lipoprotein-filled vesicles and were less heterogeneous than corresponding Golgi endosomal fractions. All endosomal fractions were enriched in receptors for insulin and prolactin but binding sites for the former were more broadly distributed in other subfractions than those for the latter. On Percoll gradient centrifugation, L endosomal fractions yielded one peak (rho 1.057) corresponding to the heavier of two peaks seen in Golgi endosomal fractions. The protein composition of high density L and Golgi endosomes, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was similar. The bulk of marker enzymes assayed did not migrate with the endosomal components. Combined acid phosphatase cytochemistry and electron microscope radioautography established that about 80% of the L endosomes contained no acid phosphatase. By affinity labeling and immunological titration with insulin receptor antibody, insulin receptors were identical in L and Golgi endosomes. Insulin-stimulable receptor kinase was demonstrable in both L and Golgi endosome fractions. Following in vivo insulin administration, the insulin receptor kinase in both L and Golgi endosomes was significantly activated. This activated state was not inhibited by a large excess of antiserum to insulin and thus not due to insulin contaminating the partially purified receptor preparation. These observations are compatible with the maintenance and/or initiation of hormone-dependent phosphorylations intracellularly.