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• W2114046802 abstract "Insulin signal transduction, initiated by binding of insulin to its receptor at the plasma membrane, activates the intrinsic receptor tyrosine kinase and leads to internalization of the activated ligand-receptor complex into endosomes. This study addresses the role played by the activated insulin receptor within hepatic endosomes and provides evidence for its central role in insulin-stimulated events in vivo. Rats were treated with chloroquine, an acidotrophic agent that has been shown previously to inhibit endosomal insulin degradation, and then with insulin. Livers were removed and fractionated by density gradient centrifugation to obtain endosomal and plasma membrane preparations. Chloroquine treatment increased the amount of receptor-bound insulin in endosomes at 2 min after insulin injection by 937 as determined by exclusion from G-50 columns and by 907 as determined by polyethylene glycol precipitation (p < 0.02). Chloroquine treatment also increased the insulin receptor content of endosomes after insulin injection (integrated over 0–45 min) by 317 when compared with controls (p < 0.05). Similarly, chloroquine increased both insulin receptor phosphotyrosine content and its exogenous tyrosine kinase activity after insulin injection (647;p < 0.01 and 967 and p < 0.001, respectively). In vivo chloroquine treatment was without any observable effect on insulin binding to plasma membrane insulin receptors, nor did it augment insulin-stimulated receptor autophosphorylation or kinase activity in the plasma membrane. Concomitant with its effects on endosomal insulin receptors, chloroquine treatment augmented insulin-stimulated incorporation of glucose into glycogen in diaphragm (p < 0.001). These observations are consistent with the hypothesis that chloroquine-dependent inhibition of endosomal insulin receptor dissociation and subsequent degradation prolongs the half-life of the active endosomal receptor and potentiates insulin signaling from this compartment. Insulin signal transduction, initiated by binding of insulin to its receptor at the plasma membrane, activates the intrinsic receptor tyrosine kinase and leads to internalization of the activated ligand-receptor complex into endosomes. This study addresses the role played by the activated insulin receptor within hepatic endosomes and provides evidence for its central role in insulin-stimulated events in vivo. Rats were treated with chloroquine, an acidotrophic agent that has been shown previously to inhibit endosomal insulin degradation, and then with insulin. Livers were removed and fractionated by density gradient centrifugation to obtain endosomal and plasma membrane preparations. Chloroquine treatment increased the amount of receptor-bound insulin in endosomes at 2 min after insulin injection by 937 as determined by exclusion from G-50 columns and by 907 as determined by polyethylene glycol precipitation (p < 0.02). Chloroquine treatment also increased the insulin receptor content of endosomes after insulin injection (integrated over 0–45 min) by 317 when compared with controls (p < 0.05). Similarly, chloroquine increased both insulin receptor phosphotyrosine content and its exogenous tyrosine kinase activity after insulin injection (647;p < 0.01 and 967 and p < 0.001, respectively). In vivo chloroquine treatment was without any observable effect on insulin binding to plasma membrane insulin receptors, nor did it augment insulin-stimulated receptor autophosphorylation or kinase activity in the plasma membrane. Concomitant with its effects on endosomal insulin receptors, chloroquine treatment augmented insulin-stimulated incorporation of glucose into glycogen in diaphragm (p < 0.001). These observations are consistent with the hypothesis that chloroquine-dependent inhibition of endosomal insulin receptor dissociation and subsequent degradation prolongs the half-life of the active endosomal receptor and potentiates insulin signaling from this compartment. Insulin signal transduction is initiated by binding of insulin to its receptor at the plasma membrane, which in turn leads to the rapid autophosphorylation of multiple tyrosine residues on the intracellular portion of the ॆ-subunit and the activation of the receptor tyrosine kinase toward exogenous substrates (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Cheatham B. Kahn C.R. Endocr. Rev. 1995; 16: 117-142Crossref PubMed Google Scholar). Following autophosphorylation, the activated ligand-receptor complex is internalized into endosomes in liver (3Khan M.N. Savoie S. Bergeron J.J.M. Posner B.I. J. Biol. Chem. 1986; 261: 8462-8472Abstract Full Text PDF PubMed Google Scholar, 4Khan M.N. Baquiran G. Brule C. Burgess J. Foster B. Bergeron J.J.M. Posner B.I. J. Biol. Chem. 1989; 264: 12931-12940Abstract Full Text PDF PubMed Google Scholar, 5Burgess J.W. Wada I. Ling N. Khan M.N. Bergeron J.J.M. Posner B.I. J. Biol. Chem. 1992; 267: 10077-10086Abstract Full Text PDF PubMed Google Scholar, 6Drake P.G. Bevan A.P. Burgess J.W. Bergeron J.J.M. Posner B.I. Endocrinology. 1996; 137: 4960-4968Crossref PubMed Scopus (47) Google Scholar) and low density membranes in adipocytes (7Klein H.H. Freidenberg G.R. Matthaei S. Olefsky J.M. J. Biol. Chem. 1987; 262: 10557-10564Abstract Full Text PDF PubMed Google Scholar, 8Kublaoui B. Lee J. Pilch P.F. J. Biol. Chem. 1995; 270: 59-65Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and muscle (9Eckel J. Reinauer H. Biochem. J. 1988; 249: 111-116Crossref PubMed Scopus (11) Google Scholar). Endocytosis of activated receptors has the twin effects of concentrating receptors within endosomes and allowing the insulin receptor tyrosine kinase to phosphorylate substrates that are spatio-temporally distinct from the plasma membrane (Ref. 10Bevan A.P. Burgess J.W. Drake P.G. Shaver A. Bergeron J.J.M. Posner B.I. J. Biol. Chem. 1995; 270: 10784-10791Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar; reviewed in Ref. 11Bevan A.P. Drake P.G. Bergeron J.J.M. Posner B.I. Trends Endocrinol. Metab. 1996; 7: 13-21Abstract Full Text PDF PubMed Scopus (57) Google Scholar). Subsequent termination of signal transduction is achieved by endosomal insulin degradation (12Pease R.J. Smith G.D. Peters T.J. Biochem. J. 1985; 228: 137-146Crossref PubMed Scopus (34) Google Scholar, 13Pease R.J. Smith G.D. Peters T.J. Eur. J. Biochem. 1987; 164: 251-257Crossref PubMed Scopus (17) Google Scholar, 14Doherty J.J. Kay D.G. Lai W.H. Posner B.I. Bergeron J.J.M. J. Cell Biol. 1990; 110: 35-42Crossref PubMed Scopus (76) Google Scholar, 15Authier F. Rachubinski R.A. Posner B.I. Bergeron J.J.M. J. Biol. Chem. 1994; 269: 3010-3016Abstract Full Text PDF PubMed Google Scholar, 16Seabright P.J. Smith G.D. Biochem. J. 1996; 320: 947-956Crossref PubMed Scopus (34) Google Scholar) following dissociation of insulin from its receptor (14Doherty J.J. Kay D.G. Lai W.H. Posner B.I. Bergeron J.J.M. J. Cell Biol. 1990; 110: 35-42Crossref PubMed Scopus (76) Google Scholar, 17Desbuquois B. Lopez S. Janicot M. Burlet H. de Galle B. Fouque F. Diabete Metab. 1992; 18: 104-112PubMed Google Scholar) as the intralumenal environment of the endosome acidifies (18Murphy R.F. Powers S. Cantor C.R. J. Cell Biol. 1984; 98: 1757-1762Crossref PubMed Scopus (219) Google Scholar). This loss of the ligand-receptor complex attenuates any further ligand-driven receptor re-phosphorylation, and the receptor is dephosphorylated by extralumenal endosomally associated phosphotyrosine phosphatase(s) (5Burgess J.W. Wada I. Ling N. Khan M.N. Bergeron J.J.M. Posner B.I. J. Biol. Chem. 1992; 267: 10077-10086Abstract Full Text PDF PubMed Google Scholar,6Drake P.G. Bevan A.P. Burgess J.W. Bergeron J.J.M. Posner B.I. Endocrinology. 1996; 137: 4960-4968Crossref PubMed Scopus (47) Google Scholar, 19Faure R. Baquiran G. Bergeron J.J.M. Posner B.I. J. Biol. Chem. 1992; 267: 11215-11221Abstract Full Text PDF PubMed Google Scholar). The anti-malarial drug chloroquine has been shown to elicit a number of effects on insulin metabolism. Thus, in rats, chloroquine treatment leads to hepatic retention of intact insulin within endosomes (12Pease R.J. Smith G.D. Peters T.J. Biochem. J. 1985; 228: 137-146Crossref PubMed Scopus (34) Google Scholar, 20Dennis P.A. Aronson N.N. Arch. Biochem. Biophys. 1981; 212: 170-176Crossref PubMed Scopus (10) Google Scholar) due to inhibition of endosomal insulin degradation (13Pease R.J. Smith G.D. Peters T.J. Eur. J. Biochem. 1987; 164: 251-257Crossref PubMed Scopus (17) Google Scholar, 21Smith G.D. Christensen J.R. Rideout J.M. Peters T.J. Eur. J. Biochem. 1989; 181: 287-294Crossref PubMed Scopus (15) Google Scholar, 22Posner B.I. Patel B.A. Khan M.N. Bergeron J.J.M. J. Biol. Chem. 1982; 257: 5789-5799Abstract Full Text PDF PubMed Google Scholar). This effect does not rely solely on the acidotrophic nature of chloroquine (whereby it is accumulated within acidic vesicles neutralizing the pH; Ref. 23De Duve C.C. De Barsy T. Poole B. Trouet A. Tulkens P. Van Hoof F. Biochem. Pharmacol. 1974; 23: 2495-2531Crossref PubMed Scopus (1525) Google Scholar), as chloroquine's inhibition of insulin degradation persists in detergent disrupted endosomes (13Pease R.J. Smith G.D. Peters T.J. Eur. J. Biochem. 1987; 164: 251-257Crossref PubMed Scopus (17) Google Scholar, 21Smith G.D. Christensen J.R. Rideout J.M. Peters T.J. Eur. J. Biochem. 1989; 181: 287-294Crossref PubMed Scopus (15) Google Scholar). Kinetic analysis of the rates of endosomal insulin degradation in the absence of chloroquine show it to be a bi-exponential process where the values of the rate constants are very similar to those for the dissociation of insulin from its receptor (21Smith G.D. Christensen J.R. Rideout J.M. Peters T.J. Eur. J. Biochem. 1989; 181: 287-294Crossref PubMed Scopus (15) Google Scholar), suggesting that dissociation from the receptor is the rate-limiting step in degradation. Chloroquine results in a 2-fold decrease in the rate constant of the slow process, together with an increase in the proportion of degradation proceeding via the slow process (21Smith G.D. Christensen J.R. Rideout J.M. Peters T.J. Eur. J. Biochem. 1989; 181: 287-294Crossref PubMed Scopus (15) Google Scholar, 24Christensen J.R. Smith G.D. Peters T.J. Biochem. Soc. Trans. 1987; 16: 574-575Crossref Scopus (1) Google Scholar). Additionally, chloroquine has been shown to increase insulin binding in cultured hepatoma cells (25Sorimachi K. Okayasu T. Yasumura Y. Endocr. Res. 1987; 13: 49-60Crossref PubMed Google Scholar, 26de Vries C.P. Van Haeften T.W. Rao B.R. Van der Veen E.A. Diabetes Metab. 1990; 16: 70-76PubMed Google Scholar) and IM9 lymphocytes (27Iwamoto Y. Maddux B. Goldfine I.D. Biochem. Biophys. Res. Commun. 1981; 103: 863-871Crossref PubMed Scopus (6) Google Scholar). Modeling of insulin binding to purified plasma membranes in the presence of chloroquine demonstrated that the augmentation of binding was due to a decrease in the rate constant for insulin dissociation (28Bevan A.P. Christensen J.R. Tikerpae J. Smith G.D. Biochem. J. 1995; 311: 787-795Crossref PubMed Scopus (18) Google Scholar). Clinical manifestations of chloroquine action have also been observed. Thus, in non-insulin-dependent diabetes mellitus, chloroquine improves glucose tolerance (29Smith G.D. Amos T.A.S. Mahler R. Peters T.J. Br. Med. J. 1987; 294: 465-467Crossref PubMed Scopus (56) Google Scholar), increases peripheral glucose disposal, and decreases the metabolic clearance rate of insulin (30Powrie J.K. Smith G.D. Shojaee-Moradie F. Sonksen P.H. Jones R.H. Am. J. Physiol. 1991; 260: E897-E904Crossref PubMed Google Scholar). In insulin-dependent diabetes mellitus, chloroquine has been shown to reduce insulin resistance (31Blazar B.R. Whitlet C.B. Kitabchi A.E. Tsai M.Y. Santiago J. White N. Stentz F.B. Brown D.M. Diabetes. 1984; 33: 1133-1137Crossref PubMed Scopus (51) Google Scholar). This augmentation of insulin-receptor interaction observed with chloroquine treatment prompted the present study to ascertain whetherin vivo chloroquine treatment in the rat: 1) increases the amount of insulin bound to endosomal insulin receptors, 2) inhibits dissociation and hence degradation of insulin within endosomes, 3) augments and/or prolongs the autophosphorylation state and activity of the endosomal insulin receptor tyrosine kinase, 4) affects the temporal flux of insulin receptors through the endosome compartment, and 5) augments insulin-stimulated metabolic events. Male Sprague-Dawley rats 1All animal studies herein cited conformed to the Home Office Animals Act of 1986 and University of Cambridge procedure guidelines. (180–200 g, body weight) were fed ad libitum on a standard chow diet and housed at 22 ± 1 °C with 12-h light cycles. Porcine insulin suspension was purchased from Calbiochem (Notts, UK). Chloroquine (7-chloro-4-(4-diethylamino-1-methylbutylamino)quinoline), RIA 2The abbreviations used are: RIA, radioimmunoassay; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; BSA, bovine serum albumin; NEM, N-ethylmaleimide, PBS, phosphate-buffered saline; PEG, polyethylene glycol; HPLC, high performance liquid chromatography; ANOVA, analysis of variance. -grade BSA and most other chemicals were purchased from Sigma (Poole, Dorset, UK). Carrier-free Na125I and [γ-32P]ATP (9 TBq/ॖmol) were from Amersham International (Bucks., UK). Metafane anesthetic was from C-VET Ltd. (Bury St. Edmonds, Suffolk, UK), and Sagital (sodium pentabarbitone) was from May and Baker Ltd. (Dagenham, UK). Polyethylene glycol 6000 (PEG-6000; molecular weight 6000–7500) was from BDH (Poole, Dorset, UK). The synthetic peptide FYF (RDIFEADYFRK) was synthesized commercially and corresponds to residues based on the kinase regulatory domain of the insulin receptor (32Ullrich A. Bell J.R. Chen E.Y. Herrera R. Petruzzelli L.M. Dull T.J. Gray A. Coussens L. Liao Y.-C. Tsubokawa M. Mason A. Seeburg P.H. Grunfeld C. Rosen O.M. Ramachandran J. Nature. 1985; 313: 756-761Crossref PubMed Scopus (1500) Google Scholar) except that two tyrosines have been replaced with phenylalanine to afford a higher degree of linearity to the kinase assay. Monocomponent porcine insulin was prepared from insulin zinc suspension (British Pharmaceuticals) as outlined by Christensen et al. (33Christensen J.R. Smith G.D. Peters T.J. Cell Biochem. Funct. 1985; 3: 13-19Crossref PubMed Scopus (10) Google Scholar) and was iodinated with lactoperoxidase (34Jorgensen K.H. Larsen U.D. Diabetologia. 1980; 19: 546-554Crossref PubMed Scopus (75) Google Scholar). The 125I-[A14]insulin isomer was separated by HPLC as described previously (35Rideout J.M. Smith G.D. Lim C.K. Peters T.J. Biochem. Soc. Trans. 1985; 13: 1225-1226Crossref Google Scholar). A monoclonal phosphotyrosine antibody (4G10) used for immunocapture assays was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). A monoclonal antibody (CT-3) raised to, and reacting with, the last 43 amino acids of the insulin receptor ॆ-subunit was used for immunocapture assays. CT-3 was prepared using as antigen a GST-fusion protein containing the terminal 100 amino acids of the human insulin receptor (36Kasuya J. Li S.-L. Orr S. Siddle K. Fujita Yamaguchi Y. Biochem. Biophys. Res. Commun. 1994; 200: 777-783Crossref PubMed Scopus (6) Google Scholar) and purified as described previously (37Soos M.A. Field C.E. Lammers R. Ullrich A. Zhang B. Roth R.A. Andersen A.S. Kjeldsen T. Siddle K. J. Biol. Chem. 1992; 267: 12955-12963Abstract Full Text PDF PubMed Google Scholar). Following Metafane inhalation anesthesia, rats received an intraperitoneal injection of either 20 ॖmol/100 g body weight chloroquine in PBS, pH 7.4, or PBS alone at 2 and 1 h prior to insulin injection. Insulin (0.15 or 1.5 ॖg/100 g body weight) was administered via intrajugular injection in PBS, pH 7.4, containing 0.17 RIA-grade BSA. Animals were sacrificed after insulin injection at the times indicated in figure legends. In studies where PEG precipitation, HPLC, and gel permeation chromatography were under investigation, animals were anesthetized with an intraperitoneal injection of Sagital (60 mg/kg body weight). Following laparotomy,125I-[A14]insulin (1 MBq in 0.5 ml of PBS, 57 RIA-grade BSA) was injected via the hepatic portal vein over a period of 30 s and the liver removed after another 2 min. Following sacrifice, livers were removed rapidly and placed in ice-cold homogenization buffer. Plasma membranes were prepared as described by Bevan et al. (10Bevan A.P. Burgess J.W. Drake P.G. Shaver A. Bergeron J.J.M. Posner B.I. J. Biol. Chem. 1995; 270: 10784-10791Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and endosomes prepared as described below. Livers were minced with scissors in ice-cold homogenization buffer (10 mm HEPES buffer, pH 7.4, containing 0.25 msucrose, 1 mm MgCl2, 0.1 mmphenylmethanesulfonic fluoride, 0.2 mm AEBSF, 0.02 mm E-64, 0.02 mm pepstatin A, 2 mmNaF, and 2 mm sodium orthovanadate). All preparative procedures were performed at 4 °C in the presence of the same concentration of phosphatase/protease inhibitors and buffer with only the sucrose concentration changing as indicated. The livers were homogenized (5 ml of homogenization buffer/g of liver) in a Potter-Elvehjem homogenizer with five passes of a motorized Teflon pestle to achieve a 207 homogenate and then filtered through a 50-ॖm nylon mesh to remove fibrous and undisrupted tissue. A 10-ml aliquot of homogenate was layered onto a discontinuous gradient of 11 ml ρ = 1.09 g·cm−3 (0.67 m) and 12 ml ρ = 1.14 g·cm−3 (1.05 m) sucrose in 33-ml centrifuge tubes and centrifuged in a Sorvall AH-629 swing-out rotor (Du Pont) at 110,000 × g av (29,000 rpm, 75 min, 4 °C) in a Sorvall OTD75B centrifuge (Du Pont). Endosomes were collected at the interface of the 1.09 and 1.14 g·cm−3sucrose solutions. In studies where PEG precipitation, HPLC, and gel permeation chromatography of 125I-[A14]insulin were under investigation, endosomes were prepared in homogenization and gradient buffers containing 10 mm HEPES buffer, pH 7.4, 2.5 mm N-ethylmaleimide (NEM), 1 mm1,10-phenanthroline, and 1 mm chloroquine. The homogenization buffer to prepare samples for HPLC analysis additionally contained 60 units/ml bacitracin. Plasma membrane and endosomal fractions (0.5 ml) were solubilized in 0.5 ml of freshly prepared 2 × solubilization buffer (50 mm HEPES buffer, pH 7.4, containing 17 Triton X-100, 150 mm NaCl, 10 mm EDTA, 10 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 30 mm NaF, 0.5 mmphenylmethanesulfonic fluoride, 2.5 mm benzamidine, 1 ॖg/ml leupeptin, 1 ॖg/ml pepstatin A, and 1 ॖg/ml antipain: final concentration). After incubation for 10 min at 4 °C, the samples were clarified by centrifugation in an Eppendorf centrifuge at 13,000 × g av (12,000 rpm, 5 min, 4 °C). Tyrosine kinase activity was assessed using a modified microtiter plate immunocapture method as described previously (38Zhang B. Tavare J.M. Ellis L. Roth R.A. J. Biol. Chem. 1991; 266: 990-996Abstract Full Text PDF PubMed Google Scholar, 39Morgan D.O. Roth R.A. Endocrinology. 1985; 116: 1224-1226Crossref PubMed Scopus (14) Google Scholar). Fifty microliters of CT-3 in 20 mmNaHCO3, pH 9.6, at a concentration of 0.5 ॖg/ml was incubated for 16 h at 4 °C in 96-well F16 Maxisorb loose Nunc-immuno microwell module plates. Any unattached antibody was removed with three applications (100 ॖl) of wash buffer (50 mm HEPES buffer, pH 7.4, 150 mm NaCl, 0.17 BSA, 0.17 Triton X-100, and 0.17 Tween 20). Duplicate solubilized plasma membrane or endosome samples (200 ॖl) were added to wells and incubated for 16 h at 4 °C. The sample was then aspirated and plates washed three times with ice-cold wash buffer (100 ॖl) and 20 ॖl of kinase reaction mixture applied (130 mm HEPES, pH 7.4, 100 ॖg/ml BSA, 1 mm EGTA, 12 mmMgCl2, 0.2 ॖm FYF, and 74 KBq [γ-32P]ATP). The incubation was terminated after 20 min at room temperature by the addition of 5 ॖl of 2 m HCl.32P-Labeled peptide substrate was captured onto phosphocellulose columns (Spinzyme™, Pierce, Chester, UK) by centrifugation in an Eppendorf centrifuge at 13,000 ×g av (12,000 rpm, 0.5 min, 22 °C). The filter was washed twice with 75 mm phosphoric acid (500 ॖl) and centrifuged as before. Finally, the filter cartridge was removed and counted in 10 ml of scintillation mixture in a Canberra Packard 1500 Tri-Carb liquid scintillation analyzer. Solubilized plasma membrane and endosomes were incubated with microtiter plates as described above in the kinase assay up to the point of kinase reaction mixture addition, except that in the case of phosphotyrosine content estimation, the wells were plated with phosphotyrosine antibody 4G10 at a concentration of 1 ॖg/ml instead of CT-3 and the sample volume was 100 ॖl. Following aspiration of the sample and three washes with wash buffer, the bound antibody was incubated for 16 h at 4 °C with 50 ॖl of binding buffer (100 mm HEPES buffer, pH 7.4, 120 mmNaCl, 1 mm EDTA, 15 mm sodium acetate, 1.2 mm MgSO4, 10 mm glucose, and 17 BSA) containing 333 Bq of 125I-[A14]insulin. The wells were then aspirated and washed three times with 100 ॖl of PBS, pH 7.4, and the bound insulin released by the addition of 100 ॖl of 0.037 SDS in water for 20 min at room temperature. The 100 ॖl was removed from the wells and counted for radioactivity in an LKB 1282 γ-counter. Following hepatic portal vein injection of 125I-[A14]insulin (1 MBq in 0.5 ml of PBS, 57 RIA-grade BSA), endosomes were prepared by discontinuous sucrose gradient centrifugation as described above. The isolated endosomal population was subsequently washed free of cytosol by a 10-fold dilution in 10 mm HEPES buffer, pH 7.4 (containing 0.1m KCl, 2.5 mm NEM), and sedimented by centrifugation in a Sorvall T-865.1 rotor at 100,000 ×g av (37,000 rpm, 30 min, 4 °C). The endosomes were solubilized with a final concentration of 0.17 Triton X-100 at 4 °C for 10 min and then clarified by centrifugation in a Beckman TLA 100.2 rotor at 120,000 × g av (60,000 rpm, 10 min, 4 °C). HPLC separation of extracted endosomal contents was performed at a flow rate of 1 ml/min, in 0.1 m ammonium acetate buffer, pH 5.5, with an acetonitrile gradient. The precise changes in acetonitrile concentration required to effect the separation of degradation intermediates were as follows: 1) 0–5 min at 07, 2) 5–20-min gradient rising to 8.457, 3) instantaneous rise to 28.57, 4) 20–40-min gradient rising to 32.57. Detection of 125I was achieved by an on-line counter attached to a reverse phase HPLC Hypersil-BDS, 5 ॖm, C-18 column, 250 × 4.6 mm, fitted with an additional 10-mm guard cartridge of the same material and purchased from Shandon HPLC (Runcorn, Cheshire, UK). Endosomes containing 125I-[A14]insulin were prepared as described above from animals that had received an injection of125I-[A14]insulin (1 MBq in 0.5 ml of PBS, 57 RIA-grade BSA) via the hepatic portal vein. The endosomes were solubilized with a final concentration of 0.17 Triton X-100 at 4 °C for 10 min and then clarified by centrifugation in a Beckman TLA 100.2 rotor at 120,000 × g av (60,000 rpm, 10 min, 4 °C). A 0.25-ml aliquot of the supernatant was mixed with 0.5 ml of bovine γ-globulin (1.25 mg/ml PBS, pH 7.4) and 0.5 ml of PEG-6000 (257 in water). The mixture was left for 20 min at 4 °C and then centrifuged in a Jouan KR 422 centrifuge at 3000 ×g av (3200 rpm, 25 min, 4 °C). The pellet was then counted for radioactivity using an LKB 1282 γ-counter. A 0.5-ml aliquot of solubilized endosomes containing 125I-[A14]insulin prepared as above was applied to a Sephadex G-50 column (40 × 1 cm) equilibrated with 50 mm HEPES buffer, pH 8.0, containing 17 mm NaCl, 2.5 mm NEM, 1.0 mm 1,10-phenanthroline, 1 mm chloroquine, 0.57 BSA. Elution was achieved with the same buffer, and 1-ml fractions were counted for radioactivity using LKB 1282 γ-counter. Aliquots (250 ॖl) derived from endosomes containing125I-[A14]insulin were added to 500 ॖl of ice-cold 207 trichloroacetic acid and incubated on ice for 10 min prior to centrifugation in an Eppendorf centrifuge at 13,000 ×g av (12,000 rpm, 5 min, 4 °C). The pellet and supernatant were then counted for radioactivity using an LKB 1282 γ-counter. Animals were treated with either PBS or chloroquine as described above prior to a 0.15 ॖg/100 g body weight insulin injection via the jugular vein, which also contained [14C]glucose (46.3 KBq/100 g body weight). One hour later, the animals were sacrificed, and their diaphragms excised, rinsed in ice-cold PBS and blotted dry prior to weighing. The extent of [14C]glucose incorporation into glycogen was determined by the method of Rafaelsen et al. (40Rafaelsen O.J. Lauris V. Renold A.E. Diabetes. 1965; 14: 19-26Crossref PubMed Scopus (43) Google Scholar). Protein content was determined by a Coomassie Blue dye binding kit (Pierce) using BSA as standard. Analyses were performed using the statistical program SPSS version 7. For all data sets, it was first determined that the data was normally distributed and that the variances were equal. These criteria were satisfied in all cases allowing the use of Student's t test to analyze for significant differences. Differences in integrated response curves were tested by two-way analysis of variance (ANOVA) (41Armitage P. Berry G. Statistical Methods in Medical Research. Blackwell Scientific Publishers, Oxford1987Google Scholar). It has been shown previously that hepatic insulin degradation in vivo occurs within endosomes (12Pease R.J. Smith G.D. Peters T.J. Biochem. J. 1985; 228: 137-146Crossref PubMed Scopus (34) Google Scholar), with dissociation of insulin from the receptor being a prerequisite for this process to occur (14Doherty J.J. Kay D.G. Lai W.H. Posner B.I. Bergeron J.J.M. J. Cell Biol. 1990; 110: 35-42Crossref PubMed Scopus (76) Google Scholar, 17Desbuquois B. Lopez S. Janicot M. Burlet H. de Galle B. Fouque F. Diabete Metab. 1992; 18: 104-112PubMed Google Scholar). To determine the percentage of insulin in endosomes that is receptor-bound, rats were administered 125I-[A14]insulin via the hepatic portal vein and hepatic endosomes freshly isolated. Fig.1 shows the elution profile from a G-50 gel permeation column of material generated from solubilized hepatic endosomes. The first peak represents receptor-bound insulin, which is size-excluded from the column and elutes in the void volume. This comprised 18.57 of the total. The amount of receptor-bound insulin was confirmed by an independent method of assessment, namely PEG precipitation of the insulin-receptor complex (19.27; TableI). The remaining 807 of the insulin eluted in the second peak from the G-50 column and comprised dissociated insulin, of which 86.37 remains intact as assessed by trichloroacetic acid precipitation (Table I). Thus, after insulin injection, 2 min of in vivo processing time, and endosome preparation time, 807 of endocytosed insulin has already dissociated from its receptor and is accessible for intra-endosomal degradation.Table IEffect of chloroquine on insulin-receptor interactions and inhibition of endosomal insulin degradation in vivoTreatmentTrichloroacetic acid precipitabilityPEG precipitabilitySepharose G-50 chromatography777(−) Inhibitors86.3 ± 5.519.2 ± 2.718.5(−) Chloroquine(+) Inhibitors97.5 ± 0.427.7 ± 1.629.7(−) Chloroquine(+) Inhibitors98.9 ± 0.252.6 ± 4.1*57.3(+) ChloroquineAnesthetized rats were injected with approximately 1 MBq of125I-[A14]insulin via the hepatic portal vein over 30 s. In some instances, chloroquine had been administered intraperitoneally 2 and 1 h prior to insulin injection ((+) chloroquine) with controls receiving PBS. The liver was removed after another 2 min and endosomes prepared as described under 舠Experimental Procedures.舡 Inhibitors of insulin degradation were present in the sucrose solutions except where indicated ((−) inhibitors). Endosomes were then disrupted and extracted into 0.17 Triton X-100 and applied to either a G-50 Sepharose column in the presence of 2.5 mm NEM, 1 mm 1,10-phenanthroline, and 1 mm chloroquine or subjected to trichloroacetic acid and PEG precipitation. Values represent the mean and S.E. from two to three separate animals. *, effect of chloroquine on the percent of insulin bound in the presence of buffer protease inhibitors (p < 0.02). Open table in a new tab Anesthetized rats were injected with approximately 1 MBq of125I-[A14]insulin via the hepatic portal vein over 30 s. In some instances, chloroquine had been administered intraperitoneally 2 and 1 h prior to insulin injection ((+) chloroquine) with controls receiving PBS. The liver was removed after another 2 min and endosomes prepared as described under 舠Experimental Procedures.舡 Inhibitors of insulin degradation were present in the sucrose solutions except where indicated ((−) inhibitors). Endosomes were then disrupted and extracted into 0.17 Triton X-100 and applied to either a G-50 Sepharose column in the presence of 2.5 mm NEM, 1 mm 1,10-phenanthroline, and 1 mm chloroquine or subjected to trichloroacetic acid and PEG precipitation. Values represent the mean and S.E. from two to three separate animals. *, effect of chloroquine on the percent of insulin bound in the presence of buffer protease inhibitors (p < 0.02). A previous study (28Bevan A.P. Christensen J.R. Tikerpae J. Smith G.D. Biochem. J. 1995; 311: 787-795Crossref PubMed Scopus (18) Google Scholar) had shown chloroquine capable of enhancing the association of insulin to its receptor. The effect of chloroquinein vitro was examined on insulin-receptor dissociation and degradation. Hepatic endosomes, containing125I-[A14]insulin internalized over a 2-min time period, were incubated for 1 h at 25 °C with concentrations of chloroquine ranging from 0.5 to 5 mm. In the absence of chloroquine, a 1-" @default.
• W2114046802 created "2016-06-24" @default.
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• W2114046802 date "1997-10-01" @default.
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• W2114046802 title "Chloroquine Extends the Lifetime of the Activated Insulin Receptor Complex in Endosomes" @default.
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