Effect of Inhibiting Vacuolar Acidification on Insulin Signaling in Hepatocytes
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m311493200
ISSN1083-351X
AutoresAlejandro Balbis, Gerardo Baquiran, Víctor Dumas, Barry I. Posner,
Tópico(s)Sphingolipid Metabolism and Signaling
ResumoPrevious studies have shown that the endosomal apparatus plays an important role in insulin signaling. Inhibition of endosomal acidification leads to a decrease in insulin-insulin receptor kinase (IRK) dissociation and insulin degradation. Thus, vacuolar pH could function as a modulator of insulin signaling in endosomes. In the present study we show that in primary hepatocytes pretreated with bafilomycin, there is an inhibition of vacuolar acidification. Incubation of these cells with insulin was followed by an augmentation of IRK activity but an inhibition of phosphatidylinositol 3-kinase/Akt activity and a decrease in insulin-induced DNA and glycogen synthesis. Bafilomycin treatment inhibited IRK recycling to the plasma membrane without affecting IRK internalization. Impaired IRK recycling correlated with a decrease in insulin signaling. We suggest that inhibiting vacuolar acidification sequesters activated IRKs in an intracellular compartment(s) where signaling is inhibited. This implies that endosomal receptor trafficking plays a role in regulating signal transduction. Previous studies have shown that the endosomal apparatus plays an important role in insulin signaling. Inhibition of endosomal acidification leads to a decrease in insulin-insulin receptor kinase (IRK) dissociation and insulin degradation. Thus, vacuolar pH could function as a modulator of insulin signaling in endosomes. In the present study we show that in primary hepatocytes pretreated with bafilomycin, there is an inhibition of vacuolar acidification. Incubation of these cells with insulin was followed by an augmentation of IRK activity but an inhibition of phosphatidylinositol 3-kinase/Akt activity and a decrease in insulin-induced DNA and glycogen synthesis. Bafilomycin treatment inhibited IRK recycling to the plasma membrane without affecting IRK internalization. Impaired IRK recycling correlated with a decrease in insulin signaling. We suggest that inhibiting vacuolar acidification sequesters activated IRKs in an intracellular compartment(s) where signaling is inhibited. This implies that endosomal receptor trafficking plays a role in regulating signal transduction. Insulin binding to its cell surface receptor (IRK) 1The abbreviation used are: IRK, insulin receptor kinase; IRS, insulin receptor substrate; PM, plasma membranes; PI, phosphatidylinositol; PBS, phosphate-buffered saline; IGF-1, insulin-like growth factor 1; DAMP, 3-(2,4-dimitroanalino)-3′-amino-N-methyldipropylamine. 1The abbreviation used are: IRK, insulin receptor kinase; IRS, insulin receptor substrate; PM, plasma membranes; PI, phosphatidylinositol; PBS, phosphate-buffered saline; IGF-1, insulin-like growth factor 1; DAMP, 3-(2,4-dimitroanalino)-3′-amino-N-methyldipropylamine. is followed by autophosphorylation, activation, and internalization of the IRK into endosomes (1Khan M.N. Baquiran G. Brule C. Burgess J. Foster B. Bergeron J.J. Posner B.I. J. Biol. Chem. 1989; 264: 12931-12940Abstract Full Text PDF PubMed Google Scholar). Although a proportion of IRK is targeted to lysosomes for degradation, most internalized IRK recycles to the plasma membrane (PM) (2Marshall S. J. Biol. Chem. 1985; 260: 4136-4144Abstract Full Text PDF PubMed Google Scholar, 3Drake P.G. Posner B.I. Mol. Cell Biochem. 1998; 182: 79-89Crossref PubMed Scopus (77) Google Scholar). Various studies have shown that signaling by growth factors and hormones, including insulin, occurs at both the cell surface and in endosomes (4Di Guglielmo G.M. Baass P.C. Ou W.J. Posner B.I. Bergeron J.J. EMBO J. 1994; 13: 4269-4277Crossref PubMed Scopus (299) Google Scholar, 5Bevan A.P. Burgess J.W. Drake P.G. Shaver A. Bergeron J.J. Posner B.I. J. Biol. Chem. 1995; 270: 10784-10791Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 6Ceresa B.P. Kao A.W. Santeler S.R. Pessin J.E. Mol. Cell. Biol. 1998; 18: 3862-3870Crossref PubMed Scopus (196) Google Scholar). Indeed the exclusive activation of endosomal IRK (5Bevan A.P. Burgess J.W. Drake P.G. Shaver A. Bergeron J.J. Posner B.I. J. Biol. Chem. 1995; 270: 10784-10791Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and epidermal growth factor receptor (7Wang Y. Pennock S. Chen X. Wang Z. Mol. Cell. Biol. 2002; 22: 7279-7290Crossref PubMed Scopus (214) Google Scholar) has been shown to yield substrate tyrosine phosphorylation and signal transduction. It follows that the extent and duration of cell signaling is subject to modulation by endosomal processes (3Drake P.G. Posner B.I. Mol. Cell Biochem. 1998; 182: 79-89Crossref PubMed Scopus (77) Google Scholar, 8Posner B.I. Curr. Opin. Invest. Drugs. 2003; 4: 430-434PubMed Google Scholar). It has been demonstrated that the endosomal IRK is dephosphorylated by an associated phosphotyrosine phosphatase (3Drake P.G. Posner B.I. Mol. Cell Biochem. 1998; 182: 79-89Crossref PubMed Scopus (77) Google Scholar, 9Faure R. Baquiran G. Bergeron J.J. Posner B.I. J. Biol. Chem. 1992; 267: 11215-11221Abstract Full Text PDF PubMed Google Scholar). Furthermore, a reduced intraendosomal pH (pH ≤ 6) promotes dissociation of internalized insulin-IRK complexes and degradation of insulin (10Hamel F.G. Posner B.I. Bergeron J.J. Frank B.H. Duckworth W.C. J. Biol. Chem. 1988; 263: 6703-6708Abstract Full Text PDF PubMed Google Scholar, 11Authier F. Rachubinski R.A. Posner B.I. Bergeron J.J. J. Biol. Chem. 1994; 269: 3010-3016Abstract Full Text PDF PubMed Google Scholar) by an acidic endosomal insulinase recently identified as cathepsin D (12Authier F. Metioui M. Fabrega S. Kouach M. Briand G. J. Biol. Chem. 2002; 277: 9437-9446Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The acidic pH of endosomes also effects a conformation-dependent inactivation of the IRK (13Contreres J.O. Faure R. Baquiran G. Bergeron J.J. Posner B.I. J. Biol. Chem. 1998; 273: 22007-22013Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Thus, the acidic pH of the endocytic compartment and the IRK-associated phosphotyrosine phosphatase(s) therein play fundamental roles in attenuating insulin signaling.Weak bases, proton ionophores, and inhibitors of vacuolar ATPases have been used to study the role of acidification in physiological processes (14Mellman I. Fuchs R. Helenius A. Annu. Rev. Biochem. 1986; 55: 663-700Crossref PubMed Google Scholar, 15Drose S. Altendorf K. J. Exp. Biol. 1997; 200: 1-8Crossref PubMed Google Scholar). In liver, chloroquine (a weak base) accumulates in hepatic endosomes leading to the inhibition of both the dissociation of insulin-IRK complexes and insulin degradation (16Posner B.I. Patel B.A. Khan M.N. Bergeron J.J. J. Biol. Chem. 1982; 257: 5789-5799Abstract Full Text PDF PubMed Google Scholar, 17Levy J.R. Olefsky J.M. Endocrinology. 1987; 121: 2075-2086Crossref PubMed Scopus (46) Google Scholar, 18Doherty 2nd, J.J. Kay D.G. Lai W.H. Posner B.I. Bergeron J.J. J. Cell Biol. 1990; 110: 35-42Crossref PubMed Scopus (77) Google Scholar). This suggests that inhibitors of vacuolar acidification would potentiate insulin signaling from the endosomal compartment, and there are studies consistent with this view. Thus, in rats, chloroquine treatment augmented and prolonged IRK activity in hepatic endosomes (19Bevan A.P. Krook A. Tikerpae J. Seabright P.J. Siddle K. Smith G.D. J. Biol. Chem. 1997; 272: 26833-26840Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Further, the administration of chloroquine to patients with type 2 diabetes mellitus decreased fasting plasma glucose levels, improved glucose tolerance, and increased plasma insulin levels (20Smith G.D. Amos T.A. Mahler R. Peters T.J. Br. Med. J. 1987; 294: 465-467Crossref PubMed Scopus (56) Google Scholar, 21Powrie J.K. Smith G.D. Shojaee-Moradie F. Sonksen P.H. Jones R.H. Am. J. Physiol. 1991; 260: E897-E904Crossref PubMed Google Scholar, 22Powrie J.K. Shojaee-Moradie F. Watts G.F. Smith G.D. Sonksen P.H. Jones R.H. Metabolism. 1993; 42: 415-419Abstract Full Text PDF PubMed Scopus (27) Google Scholar). However, it is not clear whether the effects of chloroquine in these patients are associated exclusively with improved insulin sensitivity as opposed to an increase in plasma insulin levels. In contrast, a previous study in adipocytes showed that chloroquine inhibited insulin-induced GLUT4 translocation to the PM (23Romanek R. Sargeant R. Paquet M.R. Gluck S. Klip A. Grinstein S. Biochem. J. 1993; 296: 321-327Crossref PubMed Scopus (20) Google Scholar). In view of the importance of this issue to an understanding of insulin signaling and potentially insulin resistance and in view of the above-noted discrepancies, we examined the role of vacuolar acidification on insulin signaling in primary rat hepatocytes.EXPERIMENTAL PROCEDURESMaterials—Porcine insulin (24.5 units/mg) was a gift from Lilly Research Laboratories (Indianapolis, IN). Collagenase was purchased from Worthington Biochemical Corp. (Freehold, NJ). Cell culture medium and antibiotics were obtained from Invitrogen, and Vitrogen-100 was obtained from Collagen Corp. (Toronto, Canada). [3H]Methylthymidine (20 Ci/mmol) was obtained from ICN Biomedicals, Inc., Canada Ltd. (Mississauga, Canada). ATP was purchased from Roche Applied Science. [U-14C]Glucose (300 mCi/mmol) and [γ-32P]ATP (3000 Ci/mmol) were purchased from PerkinElmer Life Sciences. The antibodies against Akt and phospho-Akt (Ser473) were purchased from New England Biolabs, Inc. (Mississauga, Canada). An antibody against tyrosine-phosphorylated proteins (PY99) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-p85, anti-IRS1, anti-IRS2, and anti-phospho-GSK-3 (Ser9/21) antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). An antibody raised against a peptide corresponding to residues 942–969 of the IRK β-subunit (α960) was used for Western blotting and prepared as previously described (25Burgess J.W. Wada I. Ling N. Khan M.N. Bergeron J.J. Posner B.I. J. Biol. Chem. 1992; 267: 10077-10086Abstract Full Text PDF PubMed Google Scholar). For immunoprecipitation of IRK, an antibody directed against the α-subunit was obtained from the serum of a patient with acanthosis nigricans (1Khan M.N. Baquiran G. Brule C. Burgess J. Foster B. Bergeron J.J. Posner B.I. J. Biol. Chem. 1989; 264: 12931-12940Abstract Full Text PDF PubMed Google Scholar). Protein A-Sepharose was obtained from Amersham Biosciences. Immobilon-P transfer membranes were obtained from Millipore Corp. Canada Ltd. (Mississauga, Canada). Bafilomycin A1, Chloroquine, poly(Glu4:Tyr1), and most other chemicals were purchased from Sigma.Cell Culture—Primary hepatocytes isolated from 120–140-g male Sprague-Dawley rats (Charles River Laboratories, Inc., St. Constant, Canada) by in situ liver perfusion with collagenase were plated on a collagen matrix (Vitrogen-100). Subconfluent cultures were prepared by seeding 1 × 106 cells, onto 9.6-cm2 six-well plates (Corning, Costar, Cambridge, MA) or 5 × 106 cells, onto 78-cm2 culture dishes (Starstedt Canada, St. Laurent, Canada). The cells were bathed for 24 h in seeding medium (Dulbecco's modified Eagle's medium/Ham's F-12 containing 10% fetal bovine serum, 10 mm HEPES, 20 mm NaHCO3, 500 IU/ml penicillin, and 500 μg/ml streptomycin) and then for 48 h in serum-free medium that differed from the seeding medium in that it lacked fetal bovine serum and contained 1.25 μg/ml fungizone, 0.4 mm ornithine, 2.25 μg/ml l-lactic acid, 2.5 × 10-8m selenium, and 1 × 10-8m ethanolamine. Serum-free medium was renewed before the addition of [3H]thymidine, insulin, and bafilomycin.Glycogen Synthesis—Glycogen synthesis was determined by incorporation of [U-14C]glucose into glycogen as previously described (26Urso B. Cope D.L. Kalloo-Hosein H.E. Hayward A.C. Whitehead J.P. O'Rahilly S. Siddle K. J. Biol. Chem. 1999; 274: 30864-30873Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Briefly, the hepatocytes (1×106 cells) were serum-deprived for 4 h and incubated for 2 h in serum-free medium containing 15 mm [U-14C]glucose in the presence or absence of bafilomycin A1 (100 nm) and insulin (100 nm). Incubations were stopped by three rapid washes with ice-cold PBS, and the cells were solubilized in 1 ml of 0.1 m NaOH. The samples were then boiled in the presence of 2 mg of carrier glycogen and precipitated overnight in 70% ethanol at -20 °C. After centrifugation, the precipitated glycogen was resuspended in 500 μl of water and incubated for 5 min at 70 °C. Incorporated radioactivity was determined by scintillation counting as previously described (27Mounier C. Lavoie L. Dumas V. Mohammad-Ali K. Wu J. Nantel A. Bergeron J.J. Thomas D.Y. Posner B.I. Mol. Cell. Endocrinol. 2001; 173: 15-27Crossref PubMed Scopus (33) Google Scholar)Thymidine Incorporation Assay—Subconfluent hepatocytes were plated on 9.6-cm2 six-well plates in serum-containing medium for 24 h and then in serum- and growth factor-free medium for an additional 48 h. Thymidine (5 μCi/ml), insulin, and bafilomycin A1 were added to cells as described in the figure legends. After an 18-h incubation, the cells were rinsed twice with 3 ml of cold PBS, incubated for 15 min at 4 °C in 10% trichloroacetic acid, solubilized at room temperature in 1 ml of 1 n NaOH, and then transferred to scintillation vials for counting of 3H in an LKB liquid scintillation counter as described before (24Band C.J. Mounier C. Posner B.I. Endocrinology. 1999; 140: 5626-5634Crossref PubMed Scopus (40) Google Scholar).Preparation of Cell Lysates—Primary rat hepatocytes were rinsed twice with ice-cold phosphate-buffered saline, pH 7.4, and solubilized with lysis buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 10 mm sodium pyrophosphate, 100 mm sodium fluoride, 1.5 mm MgCl2, 1 mm EGTA, 200 μm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10% glycerol, and 1% Triton X-100). The cell lysates were clarified by centrifugation at 10,000 × g for 20 min at 4 °C, and protein concentration was determined in the resulting supernatant.PI 3-Kinase Activity—Phosphotyrosine proteins were immunoprecipitated from cell lysates with an αPY antibody. The immunoprecipitates were extensively washed, and the protein A-Sepharose pellet was resuspended in 50 μl of kinase assay buffer (20 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.5 mm EGTA) containing 0.5 mg/ml phosphatidylinositol and assayed for PI 3-kinase activity as previously described (28Band C.J. Posner B.I. J. Biol. Chem. 1997; 272: 138-145Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar).Measurement of Exogenous IRK Activity—IRK from cell lysates were partially purified on wheat germ agglutinin columns as previously described (5Bevan A.P. Burgess J.W. Drake P.G. Shaver A. Bergeron J.J. Posner B.I. J. Biol. Chem. 1995; 270: 10784-10791Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Exogenous tyrosine kinase activity was determined using poly(Glu:Tyr::4:1) as substrate (25Burgess J.W. Wada I. Ling N. Khan M.N. Bergeron J.J. Posner B.I. J. Biol. Chem. 1992; 267: 10077-10086Abstract Full Text PDF PubMed Google Scholar).Determination of the Level of IRK Associated with the PM—Detection of PM-IRK was performed as described previously (29Levy-Toledano R. Caro L.H. Hindman N. Taylor S.I. Endocrinology. 1993; 133: 1803-1808Crossref PubMed Scopus (51) Google Scholar). Briefly, hepatocytes were incubated in the presence or absence of bafilomycin and insulin as noted in figure legends. Thereafter, hepatocytes were washed three times with ice-cold PBS-Ca2+-Mg2+ (PBS with 0.1 mm CaCl2 and 1 mm MgCl2, pH 7.4), and cell surface proteins were biotinylated by incubation with 0.5 mg/ml of Sulfo-NHS-LC-Biotin (Pierce) in PBS-Ca2+-Mg2+ for 30 min at 4 °C. The reaction was stopped by washing the dishes three times with PBS-Ca2+-Mg2+containing 15 mm of glycine. After biotinylation, the cell lysates were prepared as described above, and IRK was immunoprecipitated with an antibody directed against the α-subunit of the IRK. The immunoprecipitates were boiled in the presence of Laemmli buffer and subjected to SDS-PAGE. The proteins were transferred to Immobilon-P membranes and immunoblotted with α960 or streptavidin-horseradish peroxidase (Amersham Biosciences). Streptavidin binds to biotinylated proteins, allowing only the detection of IRK associated with the PM.Immunofluorescence—Acidic compartments in primary hepatocytes were visualized by immunofluorescence with the DAMP method as indicated by the manufacturer (Oxford Biomedical Research, Oxford, MI). Briefly, the primary hepatocytes were incubated with DAMP (30 μm) for 30 min, fixed in 4% paraformaldehyde, washed, and permeabilized by incubation with 0.5% Triton X-100. The fixed hepatocytes were incubated with a monoclonal anti-DNP antibody for 60 min and subsequently washed to remove unbound IgG. The cells were then incubated for 60 min with anti-mouse IgG conjugated to rhodamine and viewed under a Zeiss Axiovert 135 confocal microscope. Photographs were taken using a Zeiss LSM microsystem.Preparation of Endosomes—Primary hepatocytes from seven culture dishes (78 cm2) were homogenized in 12 ml of ice-cold sucrose buffer (5 mm Tris-HCl buffer, pH 7.4, 0.25 m sucrose, 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride, 1 mm Mg2Cl, 2 mm NaF, and 2 mm orthovanadate). The endosomes were prepared as previously described (1Khan M.N. Baquiran G. Brule C. Burgess J. Foster B. Bergeron J.J. Posner B.I. J. Biol. Chem. 1989; 264: 12931-12940Abstract Full Text PDF PubMed Google Scholar).RESULTSBafilomycin is a macrolide antibiotic that, at concentrations up to 1 μm, specifically inhibits vacuolar ATPases without the side effects induced by proton ionophores and weak bases (15Drose S. Altendorf K. J. Exp. Biol. 1997; 200: 1-8Crossref PubMed Google Scholar, 30Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (520) Google Scholar). In this study we use bafilomycin to evaluate the effect of inhibiting vacuolar acidification on insulin signaling.Effect of Bafilomycin on Vacuolar Acidification and Trafficking of IRK—First, we confirmed that bafilomycin inhibits vacuolar acidification by incubating primary hepatocytes with DAMP, a compound that accumulates in acidic vesicles. On performing this incubation, DAMP, detected by immunofluorescence (see "Experimental Procedures"), was found in granular structures in the presence or absence of insulin (Fig. 1, top panels). Preincubation with bafilomycin (100 nm for 30 min) abolished the granular fluorescence almost completely (Fig. 1, bottom panels), indicating that bafilomycin inhibited vacuolar acidification in primary hepatocytes.Previous work has shown that inhibition of vacuolar acidification interferes with receptor recycling (31Desbuquois B. Lopez S. Janicot M. Burlet H. de Galle B. Fouque F. Diabete. Metab. 1992; 18: 104-112PubMed Google Scholar, 32Johnson L.S. Dunn K.W. Pytowski B. McGraw T.E. Mol. Biol. Cell. 1993; 4: 1251-1266Crossref PubMed Scopus (170) Google Scholar). We used cell surface biotinylation to assess the effect of bafilomycin on insulin-induced IRK internalization and recycling to the cell surface. Hepatocytes were preincubated with insulin (100 nm) in the presence or absence of bafilomycin, and cell surface proteins were subsequently labeled with Sulfo-Biotin as described under "Experimental Procedures." The cells were lysed, the IRKs were immunoprecipitated from cell lysates, and both total and PM-associated IRKs were detected by immunoblotting with α960 and streptavidin, respectively. It can be seen that bafilomycin did not inhibit insulin-induced IRK internalization (Fig. 2a). Consistent with this was our observation that, following 15 min of incubation with insulin, the increase in the level of IRK in a crude endosomal preparation from hepatocytes was not significantly affected by bafilomycin (analysis by Student's t test, p > 0.05; data not shown).Fig. 2Inhibition by bafilomycin of IRK recycling but not internalization. a, IRK internalization: Primary hepatocytes were incubated with or without bafilomycin (Bafilo, 100 nm) for 30 min at 37 °C followed by the addition of insulin (100 nm) for the indicated times. The cell surface proteins were biotinylated as described under "Experimental Procedures." The cells were then lysed, and IRK was immunoprecipitated from these lysates using an antibody to the α-subunit of IRK. The immunoprecipitates were boiled in Laemmli sample buffer and subjected to SDS-PAGE. The gel proteins were transferred to Immobilon-P membranes that were probed with an anti-IRK antibody (α960) or with streptavidin-horseradish peroxidase (Strept) for detection of the total amount of IRK or PM-associated IRK, respectively. To assess specificity of immunoprecipitation, control samples were immunoprecipitated (IP) with normal IgG. The depicted experiment is one of three similar ones. b, IRK recycling. Hepatocytes were incubated with or without bafilomycin (100 nm) as above. Insulin (100 nm) or buffer was added for an additional 10 min to allow internalization of the IRK. The cells were subsequently washed three times with serum-free medium and incubated for an additional 10 min in the absence of insulin to permit recycling of IRK to the PM. The cell surface proteins were biotinylated, and total IRK and PM-associated IRK were determined as indicated above. WB, Western blotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate the effect of bafilomycin on IRK recycling, internalization of IRK was promoted by incubating hepatocytes with insulin (100 nm) for 10 min followed by washing to remove ambient insulin and further incubation for 10 min to allow recycling of internalized IRK. In control cells most internalized IRK recycled to the PM in the absence of ambient insulin, whereas in cells incubated with bafilomycin recycling of IRK to the PM was strikingly inhibited (Fig. 2b).Effect of Bafilomycin on Insulin Action in Primary Hepatocytes—To determine the effect of bafilomycin on insulin action, we measured the effect of insulin on thymidine incorporation and glycogen synthesis in hepatocytes incubated with or without bafilomycin. As is evident in Fig. 3, bafilomycin significantly inhibited the effect of insulin on both thymidine incorporation and glycogen synthesis by 40 and 60%, respectively.Fig. 3Bafilomycin inhibits insulin-stimulated DNA and glycogen synthesis. a, serum-starved hepatocytes were incubated with 5 μCi of [3H]thymidine for 18 h at 37 °C in presence or absence of bafilomycin (100 nm) and insulin (100 nm). Incorporation of [3H]thymidine into DNA was determined as described under "Experimental Procedures." The results are expressed as the means ± S.E. of three separate experiments, each performed in triplicate. #, p < 0.05 (versus insulin alone). b, serum-starved hepatocytes were incubated with 15 mm [U-14C] glucose for 2 h at 37 °C in presence or absence of bafilomycin (100 nm) and insulin (100 nm). Glycogen synthesis was determined as described under "Experimental Procedures." The results are expressed as the means ± S.E. of three separate experiments, each performed in triplicate. *, p < 0.01 (versus insulin alone).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Bafilomycin and IRK Activation—We next sought to determine whether the inhibitory effect of bafilomycin on thymidine incorporation and glycogen synthesis corresponded to an impairment in insulin signaling. Pretreatment of hepatocytes with bafilomycin did not modify insulin-induced augmentation of IRK phosphotyrosine content at 2 or 15 min after insulin (Fig. 4a). However, preincubation with bafilomycin did augment IRK activity significantly at 15 but not 2 min following insulin (Fig. 4b). We conclude that bafilomycin pretreatment results in an increase in IRK activity without a proportional increase in IRK phosphotyrosine content (Fig. 4c). In previous work we showed that endosomal IRK is inactivated consequent to a conformational change induced by acidic pH (13Contreres J.O. Faure R. Baquiran G. Bergeron J.J. Posner B.I. J. Biol. Chem. 1998; 273: 22007-22013Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Thus, neutralizing vacuolar pH with bafilomycin could suppress this acidification-dependent conformational change leading to increased IRK activity. This might explain the lack of correlation between IRK activity and tyrosine phosphorylation following bafilomycin treatment.Fig. 4Bafilomycin increases insulin-induced IRK activity. Hepatocytes were treated with (○) or without (•) bafilomycin (100 nm) for 30 min at 37 °C. Insulin (100 nm) or buffer was then added for an additional 2 or 15 min. IRK was subsequently partially purified from lysates using wheat germ agglutinin columns as described under "Experimental Procedures." a, aliquots of wheat germ agglutinin-purified IRK were subjected to SDS-PAGE, and the proteins were transferred to Immobilon-P membranes. The level of IRK tyrosine phosphorylation and IRK content were determined by immunoblotting using specific antibodies against phosphotyrosine proteins (αPY) and IRK (α960), respectively. To quantitate the degree of tyrosine phosphorylation of the IRK, the β-subunit bands of IRK and their phosphotyrosine content were determined by scanning densitometry and expressed as a PY/IRK ratio. Each point is the mean ± S.E. of three separate experiments. b, IRK tyrosine kinase activity toward an exogenous substrate (poly[Glu:Tyr::4:1]) was assessed in aliquots eluted from wheat germ agglutinin columns as described under "Experimental Procedures." Kinase activity is expressed per IRK content. Each point represents the mean ± S.E. of three separate experiments. *, p < 0.001 (versus 15 min insulin). c, IRK kinase activity is expressed per IRK phosphotyrosine content. Each point represents the mean ± S.E. of three separate experiments. #, p < 0.01 (versus 15 min insulin). PY, phosphotyrosine.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Bafilomycin and Downstream Signaling Events—We investigated the effect of bafilomycin on insulin-induced downstream signaling events. Hepatocytes were pretreated with or without bafilomycin, and the tyrosine phosphorylation of both IRS1 and IRS2 was assessed at 2 and 15 min after insulin. Bafilomycin had no effect on insulin-augmented IRS1 tyrosine phosphorylation (Fig. 5a, left panel) but significantly inhibited IRS2 phosphorylation by 35% (p < 0.005 versus insulin alone) in hepatocytes stimulated with insulin for 15 min (Fig. 5a, right panel).Fig. 5Bafilomycin inhibits insulin-induced tyrosine phosphorylation of IRS2 but not its association with p85. Primary hepatocytes were treated with bafilomycin (100 nm) for 30 min at 37 °C followed by insulin (100 nm) or buffer for 2 or 15 min. IRS1 and IRS2 were immunoprecipitated (IP) from lysates (1 mg of protein) with specific antibodies bound to protein-A. The immunoprecipitates were washed, boiled in Laemmli buffer, and subjected to SDS-PAGE, and the proteins were transferred to Immobilon-P membranes as described under "Experimental Procedures." The level of phosphotyrosine (a) and p85 (b) associated with IRS1 and IRS2 were determined by immunoblotting with αPY and αp85, respectively. A representative immunoblot at the top of each bar graph is shown. The results are the means ± S.E. of three to five separate experiments. *, p < 0.005 (versus 15 min of insulin). PY, phosphotyrosine; WB, Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In response to insulin, tyrosine phosphorylated IRS1 and IRS2 bind p85 (a regulatory subunit of PI 3-kinase), leading to the activation of PI 3-kinase. We thus assessed the effect of bafilomycin pretreatment on the extent of association of p85 with IRS1 and IRS2 following insulin treatment. As seen in Fig. 5b, incubation of hepatocytes with insulin stimulated the association of p85 with both IRS1 and IRS2 immunoprecipitates. Pretreatment with bafilomycin had no effect on the extent of the insulin-induced association (Fig. 5b). We then determined whether insulin-stimulated activation of PI 3-kinase associated with IRS1 or IRS2 is affected by bafilomycin. As seen in Fig. 6a, bafilomycin had no effect on PI 3-kinase activation assessed in anti-IRS1 and -IRS2 immunoprecipitates at 2 min but reduced this activity by 80% in anti-IRS2 immunoprecipitates at 15 min following incubation with insulin. Next we investigated the impact of reduced IRS2 associated PI 3-kinase activity on the total activity. Bafilomycin had no effect on PI 3-kinase activation assessed in anti-phosphotyrosine immunoprecipitates at 2 min but reduced this activity significantly by 60% at 15 min (p < 0.01 versus insulin alone) following incubation with insulin. As seen in Fig. 6c, a broad band at 160–180 kDa represents the main phosphotyrosine protein co-immunoprecipitated with p85 in response to insulin. Previous studies in hepatocytes have shown that this band corresponds almost exclusively to IRS1 and IRS2 (33Rother K.I. Imai Y. Caruso M. Beguinot F. Formisano P. Accili D. J. Biol. Chem. 1998; 273: 17491-17497Abstract Full Text Full Tex
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