Original Articles Sustained Activation of Insulin Receptors Internalized in GLUT4 Vesicles of Insulin-Stimulated Skeletal Muscle Luce Dombrowski, Robert Faure, and André Marette

Exposure of target cells to insulin results in the for- mation of ligand complexes on the cell surface and their subsequent internalization into the endosomal ne of the major metabolic actions of insulin is apparatus. A current view is that endocytosis of the to stimulate glucose transport and storage in (IR) kinase results in its rapid deacti- insulin-responsive tissues such as cardiac and vation and sorting of the IR back to the cell surface or skeletal muscles and white and brown fat. to late endocytic compartments. We report herein that, O in skeletal muscle, in vivo stimulation with insulin Insulin stimulates glucose transport mainly by inducing the induced a rapid internalization of the IR to an insulin- translocation of GLUT4 transporters from an intracellular sensitive GLUT4-enriched intracellular membrane frac- compartment to the plasma membrane (PM) (1–4) and the tion. After 30 min of stimulation, IR content and tyro- T-tubules (TTs) in skeletal muscle (5,6). The first step in this sine phosphorylation were increased by three and nine stimulation is the binding of insulin to its insulin receptor times in that fraction, respectively, compared with (IR) -subunits, autophosphorylation of the transmem- unstimulated muscles. In vitro autophosphorylation brane -subunits, and intrinsic activation of the IR tyrosine assays revealed that the kinase activity of internalized kinase activity. The IR increases tyrosine phosphorylation IRs was markedly augmented (eight to nine times) by of insulin receptor substrate (IRS) 1 and IRS-2 (7,8), which insulin. In marked contrast with hepatic endosomes or in turn activate phosphatidylinositol (PI) 3-kinase, a critical adipocyte low-density microsomes, no IR tyrosine dephosphorylation activity was observed in GLUT4- step for stimulation of GLUT4 translocation and glucose enriched vesicles isolated from skeletal muscle. The transport (9). activated IR was recovered in immunopurified GLUT4 Although much progress has been made in understanding vesicles after insulin stimulation. Insulin also the events leading to activation of GLUT4 translocation and increased tyrosine-phosphorylated insulin receptor glucose transport by insulin, the possible role of internal- substrate 1 and phosphatidylinositol 3-kinase adapter ized IR in the mediation of insulin’s metabolic effects is still (p85) subunit contents in the intracellular membrane not clear. The internalization of the IR is important for degra- fraction, but these signaling molecules were not dation of the ligand and termination of the insulin signal directly associated with GLUT4 vesicles. These results (10,11). However, accumulating evidence suggests that the show that, in skeletal muscle, the activated IR reaches internalized IR also participates in propagating insulin sig- a GLUT4-enriched compartment where its activity is apparently sustained. We propose that compartmental- naling to the endosomes and other intracellular organelles. ization of activated IRs to GLUT4 vesicles may play a Previous studies have shown that, in the liver, insulin causes role in sustaining insulin signaling at this locus in skele- a rapid internalization of activated IRs into endosomes tal muscle. Diabetes 49:1772–1782, 2000 (12–14). In adipose cells, insulin induces a rapid translocation of its own receptor from the PM to a Golgi-enriched low-den- sity microsome (LDM) membrane fraction that also contains GLUT4 (15–17). Moreover, studies in both adipocytes and liver have shown that intracellular or endosomal IRs are five to nine From the Department of Physiology and the Lipid Research Unit (L.D., times more tyrosine phosphorylated than the IR in the PM A.M.) and the Departments of Medicine and Pediatrics (R.F.), Laval Uni- versity Hospital Research Center, Ste-Foy, Québec, Canada. (10,12,13,18) and that the phosphorylation state of IRS-1 mir- Address correspondence and reprint requests to André Marette, Lipid rors that of the internalized IR in adipocytes (17). Interestingly, Research Unit, Laval University Hospital Research Center, 2705 Laurier the intracellular (endosomal) receptors are most highly Blvd., Ste-Foy, Québec, Canada, G1V 4G2. E-mail: andre.marette@crchul. ulaval.ca. phosphorylated in the tyrosine kinase and in the juxtamem- Received for publication 12 November 1999 and accepted in revised brane domains, which further supports a role for internalized form 14 July 2000. IRs in mediating insulin signaling (18). Further evidence for BSA, bovine serum albumin; EGFR, epidermal growth factor receptor; IP, immune pellet; IR, insulin receptor; IRAP, insulin-responsive aminopep- an important role of internalized IR complexes in insulin sig- tidase; IRS, insulin receptor substrate; LDM, low-density microsome; L-IM, naling also came from the use of an in vivo model in which LiBr-releasable intracellular membranes; NFM, nonfat milk; PBS, phos- endosomally located IRs are exclusively activated by the phate-buffered saline; PI, phosphatidylinositol; PIS, preimmune serum; tyrosine phosphatase (PTP) inhibitor bpV(phen), PM, plasma membrane; PMSF, phenylmethylsulfonyl fluoride; PTP, pro- tein tyrosine phosphatase; PTPase, phosphotyrosine phosphatase; TfR, which leads to phosphorylation of IRS-1 in the liver and ; TT, T-tubule. hypoglycemia (14).

1772 DIABETES, VOL. 49, NOVEMBER 2000 L. DOMBROWSKI, R. FAURE, AND A. MARETTE

However, few studies have addressed the role of internal- corrected for nonspecific binding as determined from the amount of 125I-insulin ized IRs in mediating insulin action in skeletal muscle, which or 125I-transferrin bound in the presence of an excess concentration of unlabeled is the main site of insulin-stimulated glucose disposal. In one insulin (36 nmol/l) or unlabeled transferrin (7 µmol/l), respectively. IR autophosphorylation assay. Muscle L-IM IR autophosphorylation was study, insulin was found to redistribute the IR to an intracel- measured as previously described (28) with minor modifications. Aliquots lular compartment isolated from rat muscle (19). However, the (70 µl, 50–200 µg) of L-IM membranes isolated from tissues of control or activation state of the IR and the nature of the compartment insulin-stimulated rats were preincubated on ice for 5 min with 20 µl buffer 1 in which it was internalized were not directly addressed in that (250 mmol/l HEPES, pH 7.4, 50 mmol/l MnCl2, 1.5 mmol/l dithiothreitol, 50 µmol/l Na3VO4, and 0.5% bovine serum albumin [BSA]) and then incubated study. Thus, the principal goal of the present study was to for 15 min on ice with 10 µl [-32P]ATP solution (100 µl [-32P]ATP + 25 µl 1.25-mmol/l determine whether acute insulin stimulation induces the ATP in 50 mmol/l HEPES, pH 7.4). Membranes were solubilized on ice for internalization and activation of the IR to GLUT4-enriched 60 min with 50 µl buffer 2 (150 mmol/l HEPES, pH 7.4, 3 mmol/l benzamidine, vesicles in rat skeletal muscle. We found that insulin induces 120 mmol/l sodium fluoride, 0.099% SDS, 0.3% NP-40, 4.5% Triton X-100, 2% a rapid and persistent internalization of activated IRs in intra- sodium deoxycholate, 150 mmol/l EDTA, 30 mmol/l sodium pyrophosphate, 6 mmol/l sodium orthovanadate, 3% BSA, and 3 mmol/l PMSF). IRs were then cellular GLUT4 vesicles in insulin-stimulated muscle. The immunoprecipitated by incubating the solubilized samples overnight with sustained activation of the IR is correlated with a lack of rotation at 4°C with 280 µl buffer 3 (50 mmol/l HEPES, pH 7.4, 1 mmol/l ben- PTP activity in this compartment. zamidine, 40 mmol/l sodium fluoride, 10 mmol/l sodium pyrophosphate, 2 mmol/l sodium orthovanadate, and 1 mmol/l PMSF) and 20 µl human IR anti- RESEARCH DESIGN AND METHODS body followed by 30 µl protein A agarose (Boehringer Mannheim, Laval, Québec, Canada) for an additional 45-min incubation at 4°C. Immunoprecip- Experimental protocol. This study was approved by the Animal Care and itates were concentrated by centrifugation for 3 min at high speed. The resul- Handling Committee of Laval University. Male Sprague-Dawley rats weighing tant pellets were washed once with buffer A (0.05 mol/l HEPES, pH 7.4, 1% Tri- 240–270 g were obtained from Charles River (Montreal, Canada). After an ton X-100, 1% SDS) and twice with buffer B (0.05 mol/l HEPES, pH 7.4, 0.1% overnight fast, rats were anesthetized by injection of ketamine and xylazine Triton X-100), resuspended in Laemmli sample buffer, boiled for 5 min, and (90 and 10 mg/kg i.p., respectively) before an insulin injection (Humulin R [Lilly, then subjected to SDS-PAGE. After electrophoresis, the gel was fixed for Indianapolis, IN], 8 U/kg i.v.) as previously described (5). The control group 15 min in 30% MeOH, 10% glycine, and 7.5% acetic acid; dried in a gel dryer was injected with saline only. At 1, 4, or 30 min after insulin injection, the rats (model 583; Bio-Rad, Hercules, CA); and subjected to autoradiography. To were killed, and hindlimb muscles, epididymal and perirenal adipose tissues, specifically detect phosphotyrosines, dried gels were soaked in water, and livers were rapidly removed. detached rapidly from the Whatman paper, and fixed for 30 min at room tem- Subcellular fractionation of skeletal muscle. Muscles (~7–9 g) were perature in 10% glutaraldehyde. The gel was then steeped in a 1N KOH solu- minced in buffer A (10 mmol/l NaHCO , pH 7.0, 0.25 mol/l sucrose, 5 mmol/l 3 tion in a bath at 55°C for 2 h, rinsed, and incubated for 15 min at room tem- NaN , 100 µmol/l phenylmethylsulfonyl fluoride (PMSF), 10 mmol/l sodium 3 perature with a 50% methanol solution. The gel was dried and subjected to pyrophosphate, 2 mmol/l sodium orthovanadate, and 10 mmol/l sodium fluo- autoradiography. ride). The minced muscles were then subjected to subcellular fractionation as In time-course experiments, aliquots (75–200 µg) of muscle L-IM mem- previously described in detail (5). In brief, this technique allows the simulta- branes, adipose tissue LDMs, or liver endosomes from insulin-stimulated rats neous isolation of PMs and TTs in distinct fractions from the same original mus- were preincubated on ice for 5 min with 20 µl buffer 1 and then were incubated cle sample. After several differential centrifugation steps, the PM and TT- at 37°C for up to 30 min. The reaction was stopped at different times (0, 2, 5, enriched fractions (TT and triad) are purified on discontinuous sucrose den- 10, or 30 min) by the addition of 50 µl ice-cold buffer 2, and membranes were sity gradients. The triad fraction is mostly enriched with triadic junctional TTs solubilized on ice for 60 min. In some experiments, liver endosomes were (i.e., TTs associated with cisternal sarcoplasmic reticulum vesicles) (5,20). The resuspended in HEPES-sucrose buffer (50 mmol/l HEPES, pH 7.4, 250 mmol/l procedure also yields a GLUT4-enriched intracellular membrane fraction sucrose, 1 mmol/l benzamidine, 2 mmol/l sodium fluoride, 1 mmol/l MnCl , devoid of plasma or tubular membrane markers. This LiBr-releasable intra- 2 1 mmol/l sodium orthovanadate, 1 mmol/l PMSF) containing 0.5 mmol/l LiBr, cellular membrane (L-IM) fraction is recovered after strong salt (LiBr) treat- mixed for 4 h at 4°C, pelleted by centrifugation (20 min at 280,000g), and resus- ment of the muscle homogenate early in the fractionation procedure and is pended in HEPES-sucrose buffer before measurement of the time course of purified on a 10–40% discontinuous sucrose gradient. The membrane fractions IR autophosphorylation. have been extensively characterized with immunological and enzymatic Immunoadsorption of GLUT4 vesicles. For each sample, 80 µl Dynabeads markers, and we previously demonstrated that insulin treatment induces the M-280 sheep anti-rabbit IgG (Dynal, Lake Success, NY) was washed twice for translocation of GLUT4 from the L-IM fraction to both the PM and the TTs 5 min with 400 µl phosphate-buffered saline (PBS) (pH 7.4) containing 0.1% (5,20,21). All membrane fractions were resuspended in buffer A and kept at nonfat milk (NFM) once with 0.2 mol/l Tris (pH 7.4) for 4 h at 37°C and rinsed –80°C until used. twice with PBS/NFM for 5 min at 4°C. The coated beads were coupled to the Isolation of liver endosomes and adipose tissue LDMs. Liver endosomes GLUT4 antibody as follows: for each sample, 5 µl GLUT4 polyclonal anti- were prepared as described previously (22). This endosomal fraction has body was incubated with the beads overnight at 4°C with constant rotation. been previously characterized in detail both morphologically and biochemi- The coated beads were then washed four times with 400 µl PBS/NFM to cally (22–24). GLUT4-enriched LDMs were isolated from adipose tissues as pre- remove unbound antibodies and were incubated again overnight at 4°C with viously described (25). 50 µg of the intracellular membrane fraction (L-IM) in a volume adjusted to Western blot analysis. Membranes (10 µg unless otherwise indicated) 500 µl with PBS/NFM containing protease inhibitors. The beads were washed were subjected to SDS-PAGE on a 7.5% polyacrylamide gel as described by three times with PBS/NFM containing protease inhibitors and were sepa- Laemmli (26), and Western blotting was performed as previously described (5). rated from unbound material (supernatant) by attraction to the magnet Immunoreactive bands were detected by the enhanced chemiluminescence (Dynal). The supernatant and washes were pooled and centrifuged at method. Sources for antibodies were as follows: Biogenesis (Sandown, NH) for 190,000g for 1 h. The sedimented unbound material (supernatant) and the polyclonal anti-GLUT4, Santa Cruz (Santa Cruz, CA) for polyclonal anti-annex- beads (immune pellet [IP]) were resuspended in Laemmli sample buffer and ins II and VI, Upstate Biotechnology (Lake Placid, NY) for monoclonal anti-phos- were incubated for 1 h at 37°C before SDS-PAGE and Western blot analysis. photyrosine and polyclonal anti-p85 PI 3-kinase, and Zymed (San Francisco, CA) Data analysis. Autoradiographs were analyzed by laser-scanning densitom- for monoclonal anti–transferrin receptor (TfR). Antibody to the juxtamembrane etry using a tabletop Agfa scanner (Arcus II; Agfa-Gavaert NV, Belgium) and domain (residues 942–968) of the IR (960) was prepared and purified as pre- quantified with the National Institutes of Health (Bethesda, MD) Image pro- viously described (27). The polyclonal gp160/insulin-responsive aminopeptidase gram. Data are means ± SE. The effects of the treatments were compared by (IRAP) antibody was supplied by Drs. P.F. Pilch and K.V. Kandror (Boston one-way analysis of variance. Unpaired Student’s t test was used when com- University, Boston, MA). The polyclonal anti–IRS-1 antibody was a kind gift from paring only two groups. Differences were considered to be statistically signi- Dr. C. Ronald Kahn (Joslin Diabetes Center, Boston, MA). A rabbit polyclonal ficant when P was <0.05. anti–epidermal growth factor receptor (EGFR) antibody was purchased from Calbiochem (La Jolla, CA). 125 Receptor binding assays. I-insulin (100–200 µCi/µg) was prepared using the RESULTS chloramine-T method, and insulin binding was measured using 50 µg of mem- brane as described previously (27,28). TfR binding was measured using 20 µg We assessed whether insulin induced IR internalization in of membranes according to a previously published procedure (29). All data were skeletal muscle, the main site of insulin-mediated glucose

DIABETES, VOL. 49, NOVEMBER 2000 1773 INSULIN RECEPTOR INTERNALIZATION IN GLUT4 VESICLES

disposal. Insulin (or saline) was injected in vivo for 4 or 30 GLUT4-containing L-IM fraction of insulin-stimulated muscle min, and subcellular fractions enriched in either cell surface suggests that this compartment is devoid of significant [PMs or TTs (TT and triad)] or GLUT4-enriched intracellular PTPase activity. To test this hypothesis, we compared the time membranes (L-IM) were isolated as previously described course of 32P-labeled IR autophosphorylation and deactivation (5,21). The endocytosis of IR was intense in the L-IM fraction at 37°C (28) in muscle L-IM fractions versus liver endosomes as detected both by IR immunoblotting and insulin binding and adipocyte LDMs. We determined in preliminary experi- assays (Fig. 1A, Table 1). This effect was evident 4 min after ments that peak times for in vivo insulin activation of the IR insulin injection and was further increased 30 min after the in liver endosomes, adipose tissue LDMs, and muscle L-IMs injection of insulin, a time at which the IR content almost were 2, 4, and 30 min, respectively, and these times were tripled in the intracellular fraction. A small but not significant therefore selected for measurements of IR autophosphory- decrease in the IR content was observed in the PM and TT lation. Figure 2 shows the level of alkali-resistant 32P incor- fractions after insulin stimulation. A statistically significant poration into IRs from liver endosomes (A), GLUT4-enriched and time-dependent reduction in IR content was, however, adipocyte LDMs (B), or L-IM muscle membranes (C) as a func- observed in the triad fraction of insulin-stimulated muscle, tion of time. As expected, 32P labeling in liver endosomal IRs which is enriched with junctional TTs (i.e., associated with the was maximal after 2 min and rapidly declined to 25% of the terminal cisterna of the sarcoplasmic reticulum). The smaller maximal value after 30 min. This is consistent with previously decrements in cell surface IR may be explained by the fact published values (28). 32P incorporation into IRs from that the cell surface compartments are quantitatively more adipocyte LDMs peaked at 2–5 min and also rapidly declined important than the L-IM membrane compartment. Indeed, to ~40% of maximal phosphorylation after 30 min. Similar data equal amounts of membrane proteins are compared in were obtained when LDMs were isolated from adipose tissues Fig. 1A. However, the recovery of PM and TT and triad mem- removed 30 min after insulin injection in vivo (as in muscle), branes is ~8 times greater than that of the L-IM fraction although the absolute amounts of IR associated with the (5,21). Thus, even a small decrease in IR levels in these cell LDMs were lower than those at 4 min after insulin treatment surface membrane fractions could explain the IR increments (data not shown). In stark contrast with liver endosomes or in the L-IM fraction of insulin-stimulated muscle. adipose tissue LDMs, muscle L-IM IRs remained maximally Studies in liver have shown that activated IR internalization phosphorylated even after 30 min of incubation at 37°C. is transient and that the receptor is almost totally dephos- Because the rapid dephosphorylation of activated IRs is phorylated within 5 min of insulin stimulation (22,27). Inter- believed to be mediated by membrane-associated PTPase, estingly, the results in Fig. 1A and Table 1 show that, in skele- these results strongly suggest that L-IM membranes are tal muscle, the IR remains elevated in the GLUT4-containing devoid of significant PTPase activity or that the PTPase is not intracellular fraction even 30 min after insulin injection. constitutively active in this compartment. Thus, we next determined whether the IR was still activated The redistribution of the IR between surface and intracel- in this compartment after such a long period of insulin stim- lular membranes may reflect a general effect of insulin to acti- ulation. As illustrated in Fig. 1B, the tyrosine phosphorylation vate internalization of membrane receptors into the GLUT4- of the IR was markedly increased compared with unstimu- containing L-IM fraction. To test this possibility, we have lated muscle even 30 min after insulin injection. At that time, assessed the effect of insulin on the distribution of the TfR and IR tyrosine phosphorylation was increased 8–9 times EGFR, which have been shown to recycle between the PM (Fig. 1B, left panel). When normalized for the increased and endocytic vesicles in various cell types (28,30–32). Fig- amount of IR in the L-IM fraction, the phosphorylation state ure 3A and B shows the subcellular distribution of the TfR by of the receptor was still three times more than that of unstim- immunoblotting and receptor binding assays. The TfR is ulated muscle (Fig. 1B, right panel). Thus, not only the IR con- enriched in the L-IM fraction of both stimulated and control tent but also the stoichiometry of IR tyrosine phosphorylation muscles. However, in contrast with the IR, the TfR distribu- was increased in the L-IM fraction after insulin stimulation. tion was not affected 30 min after an insulin injection. We However, the phosphorylation state of the internalized IR is found a similar lack of TfR redistribution in muscle that has not necessarily a reflection of its kinase activity (12,27). To been stimulated with insulin for only 4 min (in vivo injection), confirm that the IR tyrosine kinase activity was increased in which rules out the possibility that insulin may induce a the L-IM fraction, we measured the autophosphorylation greater effect on TfR recycling at earlier time points (data not activity of the L-IM IR in control versus insulin-stimulated shown). A lack of a significant effect of insulin on TfR recy- muscle. As seen in Fig. 1C, in vivo insulin treatment after cling is consistent with recent reports that insulin failed to 30 min increased the autophosphorylating activity of the IR induce TfR translocation (33) or only caused a small redis- eight to nine times above nonstimulated values. When cor- tribution of the receptor in skeletal muscle (34) and is con- rected for the amount of immunoprecipitated IR in the same sistent with the fact that the hormone increases the cell sur- sample, IR activity was still increased three times in the L-IM face appearance of endosomal proteins several times less than fraction, which matched the increased IR phosphorylation in GLUT4 in adipocytes (35,36). Figure 3C depicts the subcellular vivo. Taken together, these data indicate that activated IRs are distribution of another receptor kinase, the EGFR, in control rapidly internalized to a GLUT4-enriched intracellular com- and insulin-stimulated muscle. In contrast with the IR, the partment on insulin stimulation and that the activation per- EGFR was found only in the PM fraction and was unde- sists for at least 30 min. tectable in the GLUT4-enriched fraction. These data demon- That the phosphorylation state of endosomal IRs is dynam- strate that insulin-induced IR internalization and activation in ically regulated by the dephosphorylating activity of closely the GLUT4-enriched L-IM fraction are selective. associated phosphotyrosine phosphatases (PTPases) is well Taken together, the above results suggest that insulin-medi- documented (12). The sustained activation of IRs in the ated internalization of the activated IR in the insulin-respon-

1774 DIABETES, VOL. 49, NOVEMBER 2000 L. DOMBROWSKI, R. FAURE, AND A. MARETTE

A

BC (%change vs control) Autophosphorylating activity

FIG. 1. Insulin-induced IR internalization and activation in the GLUT4-enriched intracellular membrane fraction in rat skeletal muscle. Fasted male Sprague-Dawley rats weighing 240–270 g were anesthetized by intraperitoneal injection of ketamine and xylazine before an insulin (INS) (8 U/kg i.v.) or saline injection (control [CTRL]). After 4 or 30 min, the rats were killed, and hindlimb muscles were rapidly removed and then subjected to subcellular fractionation to isolate distinct fractions enriched with either PMs, TTs, triads, or L-IMs as described in RESEARCH DESIGN AND METHODS. A: Representative Western blot of the IR in the PM, TT, triad, and L-IM muscle fractions from control unstimu- lated (C) and insulin-stimulated rats (4 and 30 min). Equivalent amounts (30 µg) of proteins were subjected to SDS-PAGE on a 7.5% polya- crylamide gel and Western blotting using 960 IR antibody. Means ± SE of 4–13 experiments are shown below the representative blot. B: Effect of insulin on the tyrosine phosphorylation of the IR in the L-IM fraction. A total of 25 µg membrane proteins were subjected to SDS-PAGE and were immunoblotted with an anti-phosphotyrosine antibody. A representative gel of IR phosphotyrosine content is shown at the top, and the means ± SE of four to seven separate experiments are shown in the panel below. The right panel shows the ratio of tyrosine phosphorylation per IR in the same fraction. C: In vitro autophosphorylation (Autophosph.) of the IR in the L-IM fraction isolated from control (Ctrl) and insulin- stimulated muscle after 30 min. L-IM fractions were isolated from control and insulin-stimulated muscle after 30 min, and the autophospho- rylation assay was carried out as described in RESEARCH DESIGN AND METHODS. The amount of immunoprecipitated IR is also shown. Both the mean of IR autophosphorylation and IR autophosphorylation/IR ratio are depicted. PY, phosphotyrosine. Molecular-weight standards are shown on the right. Data are means ± SE of four to five experiments. *P < 0.05 vs. control values for all figure panels.

DIABETES, VOL. 49, NOVEMBER 2000 1775 INSULIN RECEPTOR INTERNALIZATION IN GLUT4 VESICLES

TABLE 1 Relationships between insulin-induced internalization of IR in the L-IM fraction and the translocation of GLUT4 and gp160/IRAP

IR binding IR protein content GLUT4 content IRAP content Conditions (cpm/50 µg protein) (relative densitometric) (% of control) (% of control)

Control rats 2.08 ± 0.41 278 ± 45 100 100 Insulin-injected rats 1 min 2.65 ± 0.50 366 ± 51 85 ± 9 80 ± 8 4 min 4.93 ± 0.72* 470 ± 67* 75 ± 6* 57 ± 11* 30 min 7.23 ± 2.23* 732 ± 236* 69 ± 11* 60 ± 21*

Data are means ± SE of four to six experiments except for the 1-min time point, where data are means ± SD of two individual exper- iments. L-IM membrane fractions were isolated from control rats or from rats injected with insulin for 1, 4, or 30 min as described in the legend to Fig. 1. *P < 0.05 vs. control values.

sive L-IM GLUT4 pool may be linked to GLUT4 translocation. GLUT4-depleted vesicles of the unadsorbed supernatant If this were the case, then one should expect that GLUT4 fraction and were not affected by insulin treatment. Thus, would be recruited from this compartment with a similar time these results indicate that, in skeletal muscle, activated IRs course as that of IR internalization. To test this hypothesis, we associate with intracellular GLUT4 vesicles on insulin stim- compared the effect of insulin on IR and GLUT4 content in the ulation. Our data also suggest that IRs are targeted to other L-IM fraction after 1, 4, and 30 min. As shown in Table 1, IR GLUT4-depleted membranes and/or structures, possibly internalization was already detectable only 1 min after insulin annexin II/VI–containing endocytic vesicles. stimulation (an ~30% increase) and became significant 4 min Insulin activates GLUT4 translocation by IR-mediated after insulin injection. GLUT4 content was barely decreased tyrosine phosphorylation of IRS-1, which in turn interacts with in the same compartment after 1 min (by 15%) and became the p85 adapter subunit of PI 3-kinase and activates PI notable 4 min after insulin injection. The recruitment of the 3-kinase catalytic activity. We have therefore examined insulin-responsive aminopeptidase IRAP (also known as whether these proteins are colocalized with the activated IR gp160) from the same fraction followed that of GLUT4, which in the GLUT4-enriched intracellular fraction in insulin-stim- is a finding consistent with the known coresidence of these two ulated muscle. IRS-1 was detected in the L-IM fraction, but its proteins in insulin-responsive vesicles (37,38). abundance was not increased 4 or 30 min after insulin stim- The coincidental internalization of the IR and GLUT4 ulation (data not shown) (Fig. 5A). On the other hand, IRS-1 depletion in the L-IM fraction suggests that the two phe- tyrosine phosphorylation was increased by 2- and 2.6-fold nomena are causally related. However, these proteins may be after 4 and 30 min of insulin treatment, respectively (Fig. 5A mobilized to or from different compartments within the L-IM and B). Insulin also increased the amount of p85 subunit of fraction. To verify this possibility, we have purified GLUT4 the PI 3-kinase in the L-IM fraction by 2-fold at 30 min. In con- vesicles from the L-IM fractions isolated from control and trast with the IR, however, most if not all of the tyrosine-phos- insulin-stimulated (after 30 min) muscles by immunoad- phorylated IRS-1 and PI 3-kinase adapter subunit were not sorption with GLUT4 antibody–coated magnetizable beads associated with GLUT4 vesicles but recovered in the nonad- (Fig. 4). GLUT4 vesicles were immunoadsorbed from the L- sorbed fraction of the vesicle immunopurification (Fig. 5A). IM fraction of both control and stimulated muscles [IP vs. supernatant] when using GLUT4 antibodies, but no significant DISCUSSION GLUT4 immunoisolation was observed when preimmune Several observations in the present study suggest that the IR serum (PIS) was used under identical conditions. GLUT4 is internalized in a compartment where the insulin response recoveries in the IP fraction were 78 ± 5 and 80 ± 4%, respec- is driven. We found 1) that the IR accumulates in the GLUT4- tively (n = 4, NS), for control and insulin-treated muscles. The enriched L-IM fraction even after 30 min of insulin stimulation, IR was nearly undetectable in purified GLUT4 vesicles from 2) that it was still tyrosine phosphorylated at this time, and control muscles. However, IR levels increased both in the 3) that its ability to autophosphorylate was still elevated. GLUT4 vesicles and in the unbound fraction on insulin stim- This was in marked contrast with the peak time of internal- ulation and were tyrosine phosphorylated to a similar extent. ization observed at 2–5 min after the injection of insulin in Quantification of several independent experiments revealed liver parenchyma (13,22). Furthermore, the IR is rapidly tyro- that insulin induced a marked increase in tyrosine-phospho- sine dephosphorylated in this compartment (Fig. 2) rylated IRs in association with a 33 ± 8% reduction in GLUT4 (28,41,42). This sustained IR activity in the muscle GLUT4- levels in the purified GLUT4 vesicles (Fig. 4B). In stark con- enriched compartment cannot be explained by a lack of trast, the TfR was readily detected in the GLUT4 vesicles insulin dissociation and subsequent degradation by an obtained from control muscle, and its abundance in this frac- insulin-degrading enzyme in this compartment as in the liver; tion was not significantly changed by insulin injection >90% of insulin remains receptor bound under identical con- (Fig. 4B). Annexin II (a protein believed to be involved in ditions of incubation (43). Alternatively, the GLUT4 vesicle- endosome binding and/or fusion [39]) and annexin VI (a pro- associated IR activity may not be restrained by the intralu- tein that has been suggested to be involved in receptor recy- minal acidification driven by proton pumping (44) or a puta- cling and transport to late endocytic/lysosomal compart- tive COOH-terminal inhibitory domain (12). However, given ments [40]) were both found to be mostly recovered in the the high PTPase activity observed in hepatic endosomes

1776 DIABETES, VOL. 49, NOVEMBER 2000 L. DOMBROWSKI, R. FAURE, AND A. MARETTE

FIG. 2. Time course of autophosphorylation and deactivation of 32P-labeled insulin receptors in liver endosomes (A), adipose tissue LDMs (B), and muscle L-IM fractions (C) at 37°C. L-IM muscle membranes (75–100 µg), liver endosomes (200 µg), or LDMs (100 µg) were isolated from insulin-stimulated rats and were subjected to phosphorylation with 25 µmol/l [-32P]ATP at 37°C for the indicated times. After the phospho- rylation assay, membranes were solubilized for 1 h at 4°C, and IRs were immunoprecipitated and subjected to SDS-PAGE, alkali treatment, and autoradiography as previously described (28). Bottom panels represent the means ± SE of three different experiments for muscle L-IM frac- tions. Representative time courses of two independent experiments are shown for liver endosomes and adipose tissue LDMs. Data are expressed relative to the levels of phosphorylation attained at the 2-min time point.

(28), the lack of dephosphorylation observed here cannot explain the lack of in vitro dephosphorylation observed here. be explained solely by a possible elevated tyrosine kinase This observation cannot be explained by the loss of associ- activity of the IR. An endosomal-associated PTPase that ated PTP during treatment of muscle homogenates with LiBr appears to be not active or not present in the GLUT4- (which is used for purification of the intracellular GLUT4 enriched fraction isolated from skeletal muscle would fraction) because the same concentration of LiBr did not

DIABETES, VOL. 49, NOVEMBER 2000 1777 INSULIN RECEPTOR INTERNALIZATION IN GLUT4 VESICLES Relative Densitometric Units of membrane proteins cpm/20 µg

FIG. 3. Effects of insulin on the distribution of the TfR and EGFR in membrane fractions from rat skeletal muscle. A: Representative Western blot of the TfR in the PM, TT, and L-IM fractions isolated from unstimulated control () and insulin-injected (INS) rats after 30 min (). The position of molecular-weight standards is shown on the right. Means ± SE of four to nine experiments are shown under the representative blot. B: TfR binding in the same fractions. TfR binding was measured as described in RESEARCH DESIGN AND METHODS. Data are means ± SE of four to five experiments. No significant effects of insulin were observed. C: Representative immunoblot of the EGFR and IR in PM, TT, triad, and L- IM fractions isolated from control and insulin-injected rats after 30 min. A liver endosomal fraction (EN) from a control rat is also shown. The position of molecular-weight standards is shown on the right.

affect IR dephosphorylation in hepatic endosomes in vitro reported the time course of IR activation in LDMs isolated (data not shown). Furthermore, prior detailed salt-stripping from rat adipocytes (45). This study showed that the LDM IR experiments indicated that endosome-associated PTPase was markedly activated at 8 min but declined to ~70% of its activity is tightly anchored to the membrane (28). maximal activity by 40 min. However, to our knowledge, The ability of the muscle IR to sustain its tyrosine kinase the time course of in vitro IR phosphorylation and deacti- activity may be linked to its association with a GLUT4- vation in adipocyte LDMs has not been reported yet. In the enriched vesicular compartment. Therefore, comparing IR present study, LDMs were isolated from adipose tissue activation in GLUT4-enriched vesicles isolated from muscle removed after 4 min of insulin stimulation in vivo (peak IR (L-IM) and adipose tissue (LDM) under the same conditions activation determined in preliminary experiments), and the of insulin stimulation was interesting. A previous study rate of receptor phosphorylation and dephosphorylation

1778 DIABETES, VOL. 49, NOVEMBER 2000 L. DOMBROWSKI, R. FAURE, AND A. MARETTE

FIG. 4. Immunoadsorption of GLUT4 vesicles from L-IM fractions isolated from muscles of control and insulin-stimulated (Ins) rats and effects of insulin (after 30 min) on GLUT4, IR, and IR phosphotyrosine (-PY) levels in purified GLUT4 vesicles (IP) and unbound fraction (super- natant [Sn]). GLUT4 vesicles were isolated using GLUT4 antibody–coated magnetizable beads (-GLUT4). The supernatant (unbound mater- ial) and washes were pooled and centrifuged at 190,000g for 1 h. The sedimented unbound material (supernatant) and the beads (IP) were resuspended in Laemmli sample buffer and were subjected to SDS-PAGE and Western blot analysis (A). Representative blots show IR, anti- phosphotyrosine (-PY), TfR, GLUT4, and annexin II and VI contents. Control experiments with PIS are shown on the right. The position of molecular-weight standards is shown on the right. B: The means ± SE of three to seven individual experiments.

was assessed exactly as with the muscle L-IM fraction (i.e., translocation in skeletal muscle. This is supported by the in vitro at 37°C and in the absence of insulin). We found observation of a close parallelism between the accumula- that, under these conditions, adipose tissue IR is rapidly tion of activated IRs and the departure of GLUT4 and IRAP dephosphorylated in the GLUT4-enriched LDM, which from this compartment (Table 1). Although we could not shows that the sustained activation of the IR in GLUT4 vesi- detect a significant accumulation of IRs before GLUT4 was cles is unique to skeletal muscle and that the lack of IR translocated from this compartment, this may be related to deactivation in that tissue is not linked to the presence of a the inherent difficulty in performing early time-course exper- specialized GLUT4 compartment per se. iments in vivo. In preliminary studies carried out in cultured As depicted in Fig. 6, our findings are consistent with the L6 myocytes, we found that a 1-min insulin treatment hypothesis that the activated IR is internalized and accumu- increased the amount of tyrosine-phosphorylated IRs by lates in intracellular GLUT4 vesicles and that its sustained acti- 2.7 ± 0.1–fold in the insulin-sensitive GLUT4-enriched intra- vation in this compartment promotes signaling to GLUT4 cellular compartment without any detectable transporter

DIABETES, VOL. 49, NOVEMBER 2000 1779 INSULIN RECEPTOR INTERNALIZATION IN GLUT4 VESICLES

FIG. 5. Effect of insulin (Ins) on IRS-1, IRS-1 tyrosine phosphorylation, and p85 PI 3-kinase protein levels in the muscle L-IM fraction and GLUT4 vesicles. L-IM fractions were isolated from control or insulin-stimulated rats, and GLUT4 vesicles were immunoadsorbed using GLUT4 anti- body–coated magnetizable beads (-GLUT4) as described in Fig. 4 and RESEARCH DESIGN AND METHODS. IRS-1, IRS-1 tyrosine phosphorylation (IRS-1 PY), and p85 PI 3-kinase protein levels were measured by Western blot analysis of L-IM, purified GLUT4 vesicles (IP), and unbound fraction (supernatant [Sn]). Representative blots for control and 30 min after insulin treatment are shown in A. The position of molecular-weight stan- dards is shown on the right. B: The effect of control (C) and 4 or 30 min after insulin treatment on IRS-1 phosphorylation and p85 PI 3-kinase association in the GLUT4-enriched L-IM fraction. Data are means ± SE of three to four separate experiments. *P < 0.05 vs. control values.

recruitment at this time (L.D., A.M., unpublished data). These reported that a population of GLUT4-containing recycling data support a cause-and-effect relationship between IR endosomes (based on their TfR content) is recruited to the internalization to GLUT4 vesicles and the departure of plasma membrane on contraction (but not insulin) stimula- GLUT4 from this compartment. On the other hand, insulin tion (46). Whether the internalized IRs also associate with also increased IR endocytosis to GLUT4-depleted vesicles in these contraction-responsive GLUT4-containing recycling the present study. These vesicles contain annexins and the vesicles remains unknown at present. TfR, which suggests that the IR is also internalized to endo- The exact mechanism linking the intracellular IRs and cytic vesicles and not only to GLUT4-enriched vesicles in GLUT4 translocation is still unclear. Activated IRs in the the L-IM fraction. These receptors may be engaged into the GLUT4 vesicles may bind and tyrosine phosphorylate IRS constitutive recycling of the IR within the endosomal appa- proteins, which in turn interact with the p85 adapter subunit ratus, which is important for degradation of the ligand and ter- of PI 3-kinase and activate PI 3-kinase catalytic activity (9). mination of the insulin signal (Fig. 6). We have recently Although studies in adipocytes and cultured cell lines sug-

1780 DIABETES, VOL. 49, NOVEMBER 2000 L. DOMBROWSKI, R. FAURE, AND A. MARETTE

FIG. 6. Hypothetical model of insulin-mediated internalization of activated IRs in GLUT4 vesicles from skeletal muscle and its role in GLUT4 translocation. Activated IRs are internalized to insulin-responsive GLUT4 vesicles. The IR-containing endocytic vesicles may either dock to the GLUT4 compartment (A) or dock and fuse with the GLUT4 vesicles (B). Three possible explanations exist for IR accumulation in the GLUT4 compartment: 1) the rate of IR association is greater than GLUT4 vesicle recruitment, 2) the endocytosed (endoc.) IR vesicles are physically associated (docked) with GLUT4 vesicles but are not fused with the GLUT4 compartment (docking only, A), and/or 3) GLUT4 vesicles are selec- tively budding from this compartment. Activated IRs may induce GLUT4 translocation (GLUT4 vesicle exocytosis [exoc.]) by increasing IRS- 1–dependent activation of PI 3-kinase (PI3K). Our data suggest that IR signaling is sustained in the GLUT4 vesicles because PTPase is either not present or is inactive in that compartment. Some IRs also associate with GLUT4-depleted endocytic vesicles for constitutive recycling of the receptor through the endosomal apparatus (default pathway) for degradation of the ligand and termination of the insulin signal. GLUT4 is also internalized back to GLUT4 vesicles by endocytosis, but the endocytic vesicles may be different from those used by the IR for consti- tutive recycling.

gest that IRS-1–associated PI 3-kinase is only transiently sine-phosphorylated IRs can also bind directly to the p85 activated by insulin (t1/2 ~2 min), this may not be the case adapter subunit and activate PI 3-kinase (51). Even if the for skeletal muscle. Indeed, Wojtaszewski et al. (47) fraction of PI 3-kinase activity associated with the IR is reported that IRS-1–associated PI 3-kinase activation in small, this pool of activity may fulfill a particular function muscle is sustained for up to 100 min under physiological within a subcellular compartment such as GLUT4 vesicles. hyperinsulinemia. The compartmentalization of activated Further studies will be necessary to clarify the role of IRs to GLUT4 vesicles may be involved in the sustained GLUT4 vesicle–associated IR activity in transducing the activation of insulin signaling in skeletal muscle. Our data insulin signal at this locus. are consistent with this hypothesis. Indeed, we found that IRS-1 tyrosine phosphorylation and p85 PI 3-kinase content ACKNOWLEDGMENTS are increased in the GLUT4-enriched intracellular frac- This work was supported by a grant from the Canadian Dia- tion and remained elevated for up to 30 min after insulin betes Association. L.D. was supported by a Fonds pour la stimulation. In contrast with the activated IRs, both IRS-1 Formation de Chercheurs et l’Aide à la Recherche (FCAR)- and p85 PI 3-kinase were not retained in the GLUT4 vesi- Fonds de la Recherche en Santé du Québec (FRSQ) stu- cles and were recovered in the unadsorbed fraction of the dentship. A.M. was supported by scholarships from the immunoadsorption. The lack of association of IRS-1 and Canadian Medical Research Council and the FRSQ. R.F. was p85 PI 3-kinase with GLUT4 vesicles is consistent with supported by a scholarship from the FRSQ and by the National previous observations that these signaling effectors are Science and Engineering Research Council of Canada. associated with detergent-resistant cytoskeletal elements The authors thank Suzanne Fortier and Bruno Marcotte (e.g., actin microfilaments) in adipose and skeletal muscle for expert technical assistance. We also thank Dr. Barry I. cells (48–50). Alternatively, the IR may itself drive PI-3 Posner and Dr. Paul F. Pilch for critical reading of the kinase activation independently from IRS-1. Indeed, tyro- manuscript.

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