©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Selective Activation of the Rat Hepatic Endosomal Insulin Receptor Kinase
ROLE FOR THE ENDOSOME IN INSULIN SIGNALING (*)

A. Paul Bevan (§) , James W. Burgess (¶) , Paul G. Drake (**) , Alan Shaver (1), John J. M. Bergeron (2), Barry I. Posner (§§)

From the (1) Polypeptide Hormone Laboratory, Department of Chemistry and (2) Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B2, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Insulin administration activates the insulin receptor kinase (IRK) in both plasma membrane (PM) and endosomes (ENs) raising the possibility of transmembrane signaling occurring in the endosomal compartment. Peroxovanadium compounds activate the IRK by inhibiting IR-associated phosphotyrosine phosphatase(s). Following the administration of the phosphotyrosine phosphatase inhibitor bisperoxo(1,10-phenanthroline)oxovanadate (V) anion (bpV(phen)) activation of the hepatic IRK in ENs preceded that in PM by 5 min. When colchicine treatment preceded bpV(phen) administration IRK activation in ENs was unaffected but was totally abrogated in PM. Insulin receptor substrate-1 tyrosine phosphorylation followed the kinetics of IRK activation in ENs not PM and a hypoglycemic response similar to that achieved with a pharmacological dose of insulin ensued. These studies demonstrate that ENs constitute a site for IR-mediated signal transduction.


INTRODUCTION

The insulin receptor (IR)() is a heterodimeric glycoprotein (1) consisting of two -subunits containing insulin binding sites (2, 3) and two transmembrane -subunits possessing tyrosine kinase activity in their cytosolic domains (4, 5) . Insulin binding leads to rapid tyrosine kinase activation, culminating in autophosphorylation of the -subunits (6) and internalization of the activated insulin-receptor complex into endosomes (ENs) (7, 8) . The accumulation of activated IRs in ENs is compatible with tyrosine phosphorylation of proteins in the insulin signal transduction cascade at locations topologically distinct from the PM (7, 8) . Studies utilizing insulin-mimetic agents (9, 10) , kinase inhibitory antibodies (11, 12) , and kinase impaired receptor mutants (13, 14, 15, 16) have defined the key role activation of the insulin receptor kinase (IRK) plays in realizing the biological effects of insulin (17) . Taken together these considerations support the notion that the internalized IRs are involved in insulin signal transduction.

In 1992 Faure et al.(18) reported the existence of phosphotyrosine phosphatase(s) (PTPs) in the ENs which dephosphorylate the activated autophosphorylated IR leading to attenuation of the insulin signal (18) . More recently we have described the insulin-mimetic properties of 12 crystallizable, >95% pure, peroxovanadium compounds (pVs) each containing one or two peroxo anions, an oxo anion, and an ancillary ligand (which confers stability to the complex) (19) . When administered in vivo these compounds resulted in hypoglycemia and activation of hepatic IRK (19, 20) . In contrast, activation of skeletal muscle glycogen synthesis and IRK was induced by only selected compounds (20) .

In the present study we have explored the activity of the pV compound, bisperoxo(1,10-phenanthroline)oxovanadate(v) anion (bpV(phen)). The in vivo administration of bpV(phen) results in inhibition of endosomal IR-specific PTP(s) and corresponding augmentation of endosomal IRK activity. The activation of endosomal IRK preceded and exceeded that attained in PM. Pretreatment with colchicine resulted in selective activation of the endosomal IRK by bpV(phen) accompanied by tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and hypoglycemia indicating an important role for the ENs in regulating insulin signal transduction.


EXPERIMENTAL PROCEDURES

Animals

Female Sprague-Dawley rats, 10 weeks of age (160-180 g body weight) were purchased from Charles River Canada Ltd. (St. Constant, PQ). Except where indicated animals were fasted for 16-18 h prior to experimentation.()

Materials

Porcine insulin was a gift from Eli Lilly and Co., (Indianapolis, IN). Phenylmethanesulfonic fluoride (PMSF), aprotinin, leupeptin, pepstatin A, HEPES (free acid), sodium orthovanadate, rabbit -globulin, bacitracin, TRIS, polyglutamic acid-tyrosine (4:1) (Glu:Tyr), N-acetyl-D-glucosamine, radioimmunoassay grade bovine serum albumin (BSA), colchicine, fetal and newborn calf serum, Swim's 77 media, and most other chemicals were purchased from Sigma. Wheat germ agglutinin-Sepharose 6MB (WGA-Sepharose) and Protein A-Sepharose CL-4B were from Pharmacia LKB Biotechnology (Uppsala, Sweden). Carrier-free Na[I] and -labeled [P]ATP (3000Ci/mmol) were from DuPont NEN Radiochemicals (Lachine, PQ, Canada). Adenosine 5`-triphosphate, disodium salt (ATP) was from Boehringer Mannheim (Laval, PQ, Canada). 2-Deoxy-D-[1-H]glucose (2-[H]DG; 10.6 Ci/mmol) was from Amersham International plc (Buckinghamshire, United Kingdom). Reagents for electrophoresis were from Bio-Rad with the exception of C-labeled protein standards which were supplied by Life Technologies, Inc./BRL Canada (Burlington, Ontario, Canada). Dulbecco's modified Eagle's medium and Ham's F-12 media were from Life Technologies, Inc./BRL. Penicillin, streptomycin, and fungizone were purchased from Flow Laboratories, Inc. (McLean, VA). Kodak X-Omat AR film was from Picker International (Montreal, PQ, Canada). Immobilon-P transfer membranes were from Millipore Ltd. (Mississauga, Ontario, Canada).

Preparation of Peroxovanadium Compound bpV(phen)

The compound bpV(phen) was prepared by Dr. Jesse Ng of the Department of Chemistry, McGill University as described elsewhere (19, 21) .

Hepatoma Cell Cultures

H4IIEC3 cells were a gift from Dr. D. K. Granner, Vanderbilt University. The cells were grown to confluency in Corning 150-cm flasks in Swim's 77 media supplemented with 6 mM NaHC0, 2.4 mM CaCl, 1.2 mg/ml glucose, 60 mM Tricine, 60 µM cysteine, 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml fungizone, 10 µg/ml phenol red, and 2.5% (v/v) each of fetal and newborn calf sera. The cells were placed in serum-free Dulbecco's modified Eagle's medium/Hams's F-12 for 16-20 h prior to treatment with reagents. One hour before treatment the medium was replaced with 10 ml of Kreb's Ringer bicarbonate containing 1.0% BSA. A 5-ml volume of a three times concentrated solution of bpV(phen) and/or sodium orthovanadate was then added as indicated in the text. Incubation at 37 °C was carried out for 20 min. Following the incubation, cells were washed once with ice-cold phosphate-buffered saline (PBS) and quick-frozen in ethanol-dry ice.

Solubilization and Lectin-affinity Purification of Hepatoma Cells

To partially purify cellular IRs, cells from one 150-cm flask were suspended in 24 ml of 50 mM HEPES, pH 7.6, 1 mM PMSF, 1% Triton X-100, 1 mM ammonium molybdate, 2 µg/ml benzamidine, 0.3 trypsin inhibitory units/ml aprotinin and shaken for 30 min at 4 °C. Insoluble material was removed by centrifugation at 200,000 g for 30 min and the supernatant was recycled five times through 1-ml WGA-Sepharose columns which had been equilibrated with 50 ml of 50 mM HEPES, pH 7.6, 0.1% Triton X-100, 1 mM PMSF, 1 mM ammonium molybdate, 2 µg/ml benzamidine, 150 mM NaCl. Each column was washed with 50 ml of 50 mM HEPES, pH 6.0, 10 mM MgSO, 0.1% Triton X-100, 1 mM PMSF, 1 mM ammonium molybdate, 2 µg/ml benzamidine, 150 mM NaCl followed by 20 ml of the pH 7.6 buffer. IRs were eluted from the columns with 1.2 ml of pH 7.6 buffer containing 0.3 M N-acetyl-D-glucosamine and 0.3 trypsin inhibitory units/ml aprotinin.

Antibodies

An antibody raised against a peptide corresponding to the juxtamembrane region of the IR -subunit (960) was prepared and purified on a Protein A-Sepharose CL-4B column as described previously (8) . A polyclonal IRS-1 antibody for immunoprecipitation and a monoclonal IRS-1 antibody for Western blotting (1M92-7) were kindly provided by Dr. M. F. White (Joslin Diabetes Center, Boston). A polyclonal phosphotyrosine antibody (PY) for immunoprecipitation was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). A monoclonal PY for Western blotting (P3300) was purchased from Sigma.

Injections

Following ether anesthesia rats received an intrajugular injection of either bpV(phen) in PBS or insulin in PBS containing 0.1% BSA for the times indicated. In some instances animals received an injection of colchicine in 0.9% NaCl via the same route.

Preparation of Plasma Membranes

Following intrajugular injection, animals were sacrificed by decapitation at the indicated times post-injection. The livers were exsanguinated and rapidly excised prior to mincing at scissor point in ice-cold 5 mM TRIS-HCl buffer, pH 7.4, containing 0.25 M sucrose, 1 mM benzamidine, 1 mM PMSF, 1 mM MgCl, 2 mM NaF, 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 (4 ml of 0.25 M sucrose buffer/g liver) with 10 passes of a loose fitting B pestle in a 50-ml Wheaton homogenizer. The homogenate was centrifuged at 225 g for 6 min in a JA-17 Beckman rotor to produce a supernatant (S1) and pellet (P1). S1 was retained and the P1 resuspended with three passes of the B pestle in half the original volume of 0.25 M sucrose buffer and centrifuged as before to yield P2, which was discarded, and S2 which was pooled with S1. The combined S1 and S2 were then centrifuged at 1,600 g for 10 min to yield S3 and P3. P3 was retained, resuspended in a final volume of 40 ml, and adjusted to a final sucrose concentration of 1.42 M. This was overlaid with 3 ml of 0.25 M sucrose buffer and centrifuged at 83,000 g for 1 h. The interface was removed, adjusted to a final sucrose concentration of 0.39 M and recentrifuged at 1,600 g for 10 min as before. The pellet which constituted the PM was resuspended in 0.25 M sucrose buffer.

Preparation of Endosomes

Following intrajugular injection, animals were sacrificed by decapitation at the indicated time post-injection. The livers were exsanguinated and rapidly excised prior to mincing in 0.25 M sucrose buffer. The livers were homogenized (4 ml of 0.25 M sucrose buffer/g liver) in a Potter-Elvehjem homogenizer with 6 passes of a motorized Teflon pestle at 1,500 rpm. The homogenate was centrifuged at 3,300 g for 10 min in a Beckman Ti 50.2 fixed angle rotor to yield S1 and P1. S1 was then centrifuged at 200,000 g for 40 min in the 50.2 Ti rotor to yield S2 which was discarded and P2, the microsomal fraction. This was resuspended and adjusted to a final sucrose concentration of 1.15 M and placed below 1.0 and 0.6 M sucrose solutions. Following centrifugation at 96,500 g for 205 min in a Beckman SW 28 rotor, the EN fraction at the 0.6/1.0 M interface was removed. ENs were then diluted in 0.25 M sucrose buffer and pelleted by centrifugation at 200,000 g for 40 min in the 50.2 Ti rotor. The pellet was resuspended in 0.25 M sucrose buffer.

Preparation of Cytosol

A 20% liver homogenate was prepared as described for the EN preparation and the supernatant, constituting the cytosolic fraction, was separated following centrifugation at 200,000 g for 45 min in a SW40 Beckman rotor.

Solubilization and Lectin-affinity Purification of Plasma Membrane and Endosomal Insulin Receptors

PM and ENs were suspended in 4.5 ml of 0.25 M sucrose buffer as described above but additionally containing 20 µM leupeptin, 20 µM pepstatin A, and 0.3 trypsin inhibitory units/ml aprotinin. A 0.9-ml fraction was kept for analysis and the remaining 3.6 ml was solubilized by the addition of 0.4 ml of 0.25 M sucrose buffer containing 10% Triton X-100. The fractions were solubilized for 1 h at 4 °C on an Eberbach shaker (Ann Arbor, MI) at low speed followed by centrifugation at 200,000 g for 30 min to remove insoluble material. The solubilized material was recycled five times over 2-ml WGA-Sepharose columns which had been pre-equilibrated with 80 ml of 50 mM HEPES buffer, pH 7.6, containing 150 mM NaCl, 1 mM benzamidine, 1 mM PMSF, 0.1% Triton X-100, and 2 mM sodium orthovanadate. The columns were then washed with 80 ml of 50 mM HEPES buffer, pH 6.0, containing 150 mM NaCl, 10 mM MgSO, 1 mM benzamidine, 1 mM PMSF, 0.1% Triton X-100, and 0.1 mM sodium orthovanadate to remove any insulin still bound to the receptor, followed by 20 ml of the above buffer adjusted to pH 7.6, to return the column to physiological pH. The receptor was eluted with 2 ml of the 50 mM HEPES buffer, pH 7.6, containing 0.3 M N-acetyl-D-glucosamine (elution buffer).

Insulin Binding Determination

I-Insulin was prepared using the chloramine-T method as described previously (22) and had a specific activity of 130-200 µCi/µg. Specific binding of I-insulin to intact and lectin-purified receptor was determined at three different concentrations after overnight incubation at 4 °C as described previously (8) .

Protein Assay

Protein content was determined by a modification of Bradford's method using BSA as standard (23) .

Measurement of IRK Activity

Exogenous tyrosine kinase activity was determined using poly(Glu:Tyr) as the substrate. Each assay was performed with an amount of IR which bound 10 fmol of I-insulin in the binding assay as described previously (8) .

Immunoprecipitation of WGA-purified Fractions

WGA-Sepharose purified IR (100 fmol) was diluted to a final volume of 250 µl in elution buffer. Fifty microliters of control IgG, PY, or 960 were added and incubated at 4 °C for 4 h on a shaker at low speed. Next 150 µl of a 50% slurry of Protein A-Sepharose CL-4B was added and the mixture shaken for a further 1 h. The Protein A-Sepharose CL-4B was then pelleted by centrifugation at 12,000 g in an Eppendorf microcentrifuge for 5 min at 4 °C. The supernatant was carefully removed and assayed for exogenous tyrosine kinase activity and insulin binding.

Immunoprecipitation of IRS-1

Cytosolic protein (16 mg) was incubated in the presence of Triton X-100 (1% final concentration) for 1 h at 4 °C in a volume of 1.1 ml. Any insoluble material was removed by centrifugation at 12,000 g for 5 min in an Eppendorf microcentrifuge following which 10 µg of IRS-1 in 10 µl was added and the incubation continued for a further 4 h. A 50% slurry of protein-A Sepharose (150 µl) pre-equilibrated in 50 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM sodium orthovanadate, was added, shaken for a further 1 h, centrifuged as before and the pellet rinsed with 1 ml of wash buffer (50 mM HEPES, pH 7.4; containing 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 100 mM NaF, and 2 mM sodium orthovanadate) three times followed by boiling in 210 µl of Lammeli sample buffer (2.3% SDS, 10% glycerol, 100 mM dithiothreitol, and 0.37 M Tris-HCl, pH 6.8: final concentration) for 5 min. Seventy-microliter samples were subjected to SDS-PAGE (7.5% gel) and then transferred to Immobilon-P membranes. PY and IRS-1 content of the immunoprecipitates were determined by Western blotting using P3300 and 1M92-7 antibodies, respectively, visualized by autoradiography, and quantified by laser densitometry.

Western Blotting

For analysis of PY, IR, or IRS-1 content, either equal amounts of protein or femtomole of IR binding capacity were used and 3 Lammeli sample buffer (6.9% SDS, 30% glycerol, 300 mM dithiothreitol, and 1.1 M Tris-HCl, pH 6.8) was added. The samples were boiled for 5 min and subjected to SDS-PAGE under reducing conditions before electrophoretic transfer of phosphoproteins from SDS gels to nitrocellulose membranes (8) . After transfer the membranes were blocked for 1 h (see below). The blocking solution was then exchanged for a 50-ml solution containing the primary antibody and gently shaken for 2 h at room temperature. This was followed by three 10-min washes with 50 ml of washing solution before the blots were then transferred to 50 ml of I-labeled secondary antibody solution (700,000 cpm/electrophoretic lane transferred) for 1 h at room temperature, followed by three additional washes. The nitrocellulose membranes were mounted on Whatman 3MM paper squares and allowed to air dry. Labeled proteins were visualized by autoradiography at -70 °C using enhancing screens and Kodak X-Omat AR film. For PY Western blots all incubations were in the presence of PBS containing 20% fetal calf serum and washes were in PBS containing 1% Tween 20. For IR Western blots all incubations were in the presence of PBS containing 4% powdered milk and washes were in PBS containing 1% Tween 20. For IRS-1 Western blots all procedures were in the presence of 10 mM TRIS, pH 7.4, 300 mM NaCl, and 0.05% Tween 20 (TNT) containing 3% BSA and washes were in TNT.

IR Dephosphorylation Assay

IR dephosphorylation was assessed in rat liver ENs prepared 2 min following the intrajugular injection of 1.5 µg of insulin/100 g of body weight as described previously (18) . Briefly, ENs (25 µg) were incubated at 37 °C with 25 µM [-P]ATP for 5 min. EDTA and unlabeled ATP at final concentrations of 10 and 500 µM, respectively, were then added to terminate P labeling of the IR and allow dephosphorylation to occur. After 10 min of further incubation at 37 °C, ENs were solubilized, immunoprecipitated overnight at 4 °C with IR antibody, and subjected to SDS-PAGE on a 7.5% gel. Phosphoserine and phosphothreonine were removed by KOH digestion, the gels dried, and the phosphotyrosine content of the IR visualized by autoradiography. The extent of [P] loss from the IR -subunit, measured in the presence and absence of bpV(phen), was used to calculate the percent inhibition effected by bpV(phen).

Measurement of Plasma Glucose Levels

Rats were fasted for 4 h prior to excision of the tail tip. Following a further 30 min (recovery period), 60-µl blood samples were obtained to assess basal plasma glucose values. Animals were then injected intravenously with either 0.9% saline or colchicine (25 µmol/100 g body weight) via the tail vein. One hour later PBS or bpV(phen) was injected and blood samples collected into heparinized tubes at 15-min intervals thereafter. Plasma was obtained by centrifugation at 12,000 g for 5 min (Beckman Microfuge B) and plasma glucose concentrations determined in duplicate using a glucose analyzer-2 (Beckman instruments) as previously noted (19) . Uptake of 2-Deoxy[1-H]glucose-Following the intrajugular injection of a combination of 18.75 µCi/100 g body weight 2-[H]DG with either PBS, 0.6 µmol/100 g body weight bpV(phen) or 1.5 µg/100 g body weight insulin, the animals were sacrificed at 15 min post-injection time by decapitation and the bodies exsanguinated. Diaphragm, soleus, tibialis anterior and extensor digitorum longus muscles were removed and muscle 2-[H]DG-6-phosphate content determined as described elsewhere (24) .

Statistical Analysis

Welch's test was used to analyze for significant differences between groups provided that the samples were normally distributed. If the groups differed significantly from a normal distribution then the Wilcoxon rank sum test was employed (25) .


RESULTS

Effect of Vanadate on IRK Activation and Inhibition of Endosomal PTPs by bpV(phen)

It was previously demonstrated that incubation of H4IIEC3 hepatoma cells for 20 min with 1 mM bpV(phen) produced activation of hepatoma cell IRK, whereas incubation with 1 mM vanadate was virtually ineffective (19) . Fig. 1 demonstrates that the incubation of bpV(phen) (1 mM) with increasing concentrations of vanadate (0.01-10 mM) resulted in a dose-dependent inhibition of IRK activation. In contrast, increasing concentrations of vanadate did not significantly affect the inhibitory influence of bpV(phen) on endosomal IRK dephosphorylation (Fig. 1). Together these observations suggest that vanadate competes for a common cellular transport process with bpV(phen) but is ineffective at a major site of bpV(phen) action, the inhibition of IRK dephosphorylation. They further imply that bpV(phen) action follows its entry into the cell.


Figure 1: Effect of vanadate on the activation of the IRK and inhibition of endosomal PTPs by bpV(phen). To assess the effect of vanadate on the activation of the IRK by bpV(phen), H4IIEC3 hepatoma cells were incubated in the presence of 1 mM bpV(phen) and the indicated concentration of vanadate for 20 min. The cells were then washed, solubilized, IRs partially purified by lectin chromatography, and the IRK activity measured as described under ``Experimental Procedures.'' Each point is the mean ± S.E. of two to five separate determinations. To assess the effect of vanadate on inhibition of endosomal IR tyrosine dephosphorylation, animals were injected with 1.5 µg/100 g body weight insulin for 2 min, ENs were prepared, and the PTP assay was performed in the presence of 10 µM bpV(phen) and the indicated concentrations of vanadate as described under ``Experimental Procedures.'' Each observation is the mean ± S.E. of two to five separate determinations.



IRK Activation and PTP Inhibition in ENs following the in Vivo Administration of bpV(phen) and Insulin

We previously demonstrated the accumulation of maximally activated IRK within hepatic ENs by 2 min following in vivo insulin administration (7, 8) . More recently we showed that prior treatment of rats with bpV(phen) greatly augmented insulin-induced endosomal IRK activation and that there was a significant correlation between IRK activation in hepatoma cells and the inhibition of IRK dephosphorylation in hepatic ENs by pVs (19) . In the present study we sought to examine this correlation further by comparing IRK activation and the inhibition of IRK dephosphorylation within the same hepatic endosomal system. Rats were pretreated with bpV(phen) from 5 min to 6 h and, at each time, were sacrificed 2 min after insulin injection (Fig. 2). The tyrosine kinase activity of IRKs, partially purified from ENs by WGA chromatography, was assessed using PGT as substrate. The inhibition of IRK dephosphorylation and hence of IRK-associated PTP(s) activity was measured by assessing the loss of [P] from autophosphorylated endosomal IRKs as described under ``Experimental Procedures.'' Fig. 2A shows that the in vivo administration of bpV(phen) anywhere between 5 min and 6 h before insulin administration augmented insulin-stimulated IRK activation in a time-dependent manner. Maximal activation was seen at 15-45 min with a subsequent decrease of the activation state to approximately baseline levels by 5 h after bpV(phen) injection. The time course and extent of inhibition of IRK-associated PTP activity closely followed that of IRK activation (Fig. 2 A). There was thus a strong correlation between the in vivo activation of the endosomal IRK and inhibition of the corresponding IRK-associated PTP(s) (Fig. 2 B; r = 0.97, p < 0.001).


Figure 2: Correlation between IRK activation and PTP inhibition in ENs after treatment with bpV(phen) and insulin. Rats were fasted overnight and given an intrajugular injection of 0.6 µmol/100 g body weight bpV(phen). They then received a second injection of 1.5 µg/100 g body weight insulin 2 min prior to sacrifice at the indicated times. Hepatic ENs were prepared and IRK and PTP activities measured as described under ``Experimental Procedures.'' Panel A, the time course of activation of the IRK and the corresponding inhibition of PTP activity. Panel B, the linear correlation between IRK activation and PTP inhibition at each time point. Each observation is the mean of determinations on two to five animals.



Time Course of IRK Activation and Tyrosine Phosphorylation in PM and ENs after bpV(phen)

The above observations suggest that bpV(phen) acts by entering the cell and inhibiting IRK-associated PTP(s) resulting in IRK activation. We next sought to evaluate the time course and extent of IRK activation in vivo and focused on PM and ENs, the two major subcellular compartments in rat liver harboring IRs (7, 26) . Using well-characterized methods to separate PM from ENs (7, 8) we measured IRK activation in these cell fractions by assessing IRK-mediated phosphorylation of PGT as well as the phosphotyrosine content of the IRK. Following the in vivo administration of bpV(phen) activation of the IRK in ENs was more rapid and significantly greater than that observed in PM (Fig. 3 A). There was a reproducible lag of about 5 min in IRK activation in PM, whereas no such lag was observed in ENs. In addition, 87.7% ± 3.1% (S.E.; n = 3) of the tyrosine kinase activity of lectin-purified endosomal IRK preparations (15 min after bpV(phen)) was immunoprecipitated by 960, an IRK specific antibody (8) , confirming IRK as the major tyrosine kinase activated by bpV(phen) treatment (). Furthermore, the phosphotyrosine content of the endosomal IRK was much greater than that in PM (Fig. 3 B) with a clear signal detectable for the EN, but not PM IRK by 5 min. Treatment with bpV(phen) did not produce a change in the IR content of either PM or ENs as assessed by insulin binding () or immunoblotting with 960 (data not shown). Thus, unlike the situation following insulin administration (8) the administration of bpV(phen) was not followed by the recruitment of IRKs into ENs from PM.


Figure 3: Time course of activation and phosphotyrosine content of the PM and EN IRK by bpV(phen). Rats were fasted overnight and given an intrajugular injection of 0.6 µmol/100 g body weight bpV(phen). Following sacrifice at the times indicated, hepatic PM and ENs were prepared, solubilized, and IRs partially purified by lectin chromatography as described under ``Experimental Procedures.'' Panel A, the IR content of PM () and ENs () was determined by measuring I-insulin binding and exogenous tyrosine kinase activity assessed using PGT as substrate as described under ``Experimental Procedures'' and expressed as pmol/10 min/10 fmol of insulin binding. Each point reflects the mean ± S.E. of determinations on 3-13 separate animals. Panel B, 50 fmol of insulin binding was loaded onto a 7.5% gel for SDS-PAGE. Proteins were transferred onto PVDF Millipore Immobilon-P membranes and the PY content of the IR -subunit assessed by Western blotting using a PY antibody (PY) and a I-labeled goat anti-mouse second antibody as described under ``Experimental Procedures.''



Time Course of bpV(phen)-induced IRK Activation in PM and ENs following Colchicine Pretreatment

The delay in and lower level of IRK activation in PM compared to ENs, along with the failure of IRs to redistribute after bpV(phen) administration, suggested that the primary site of IRK activation may be in ENs with changes in PM IRK reflecting recycling of IRs from ENs back to PM. We elected to employ colchicine, an inhibitor of microtubular function, to prevent receptor recycling in liver parenchyma. This was based on the known microtubule requirements for exocytosis in liver parenchyma and other mammalian cells (27, 28, 29) and the microtubule requirement for endosomal transport to the Golgi-bile canalicular region for late endosomal/lysosomal maturation in liver parenchyma (30, 31, 32) . Recently we have observed that colchicine inhibits recycling of the hepatic prolactin receptor.() We thus administered colchicine 1 h prior to bpV(phen) treatment to determine whether selective activation of endosomal IRKs occurred in this circumstance. Colchicine treatment 60 min prior to bpV(phen) administration resulted in IRK activation exclusively in ENs with the level attained being identical to that in rats not receiving colchicine pretreatment (Fig. 4 A). In this circumstance IRK tyrosine phosphorylation was seen only in ENs (Fig. 4 B) and there was little redistribution of IR content as compared to that seen with bpV(phen) alone (). As a further control we assessed the effect of colchicine treatment on insulin-stimulated activation of the hepatic IRK. Colchicine was administered to rats 60 min prior to insulin injection and IRK activity was assessed at 30 s and 2 min post-insulin in PM and ENs, respectively, the previously established times of maximal IRK activation in these compartments (8) . Following insulin administration, IRK activity (pmol/10 min/10fmol insulin binding; mean ± S.D., n = 3) in PM was 19.3 ± 3.1 without and 18.1 ± 1.5 with colchicine pretreatment ( p = 0.58); while that in ENs was 14.3 ± 2.7 without and 18.5 ± 2.7 with colchicine pretreatment ( p = 0.13). Hence colchicine did not impair IRK activation in either PM or ENs, and we thus ascribe its effect to its activity as an inhibitor of membrane recycling.


Figure 4: Time course of activation of the PM and EN IRK by bpV(phen) following colchicine treatment. Rats were fasted overnight and given an intrajugular injection of 25 µmol/100 g body weight colchicine in 0.9% saline 1 h prior to injection with 0.6 µmol/100 g body weight bpV(phen) and sacrificed at the indicated times. Hepatic PM and ENs were prepared, solubilized, and IRs partially purified by lectin chromatography as described under ``Experimental Procedures.'' Panel A, the IR content of PM () and ENs () was determined by measuring I-insulin binding and exogenous tyrosine kinase activity assessed using PGT as substrate as described under ``Experimental Procedures'' and expressed as pmol/10 min/10 fmol of insulin binding. Each point reflects the mean ± S.E. of determinations on three to four separate animals. Panel B, 20 fmol of insulin binding was loaded onto a 7.5% gel for SDS-PAGE. Proteins were transferred onto PVDF Millipore Immobilon-P membranes and the PY content of the IR -subunit assessed by Western blotting using a PY antibody (PY) and a I-labeled goat anti-mouse second antibody as described under ``Experimental Procedures.''



Phosphotyrosine Content of IRS-1 following bpV(phen) Treatment in the Presence and Absence of Colchicine

Using a combination of colchicine and bpV(phen) we have generated an in vivo system wherein only hepatic endosomal IRK was activated. In order to determine whether insulin signaling could arise from this compartment we assessed whether tyrosine phosphorylation of IRS-1 could be observed in this situation. As can be seen in Fig. 5the administration of bpV(phen) after colchicine augmented PY phosphorylation of IRS-1 in a time-dependent fashion above that observed in control animals (3-fold at 15 min ( p < 0.02) and 5-fold at 30 min ( p < 0.01)). Even in the absence of colchicine pretreatment, increased IRS-1 phosphorylation was observed 5 min after bpV(phen) when PM IRK activation was not evident (Fig. 3 A). Also of interest is the observation that bpV(phen) treatment resulted in an increase in the amount of IRS-1 present in the cytosol compared to that observed in controls (2-fold at 15 min ( p < 0.05) and 3-fold at 30 min ( p < 0.01)). The possibility that IRS-1 phosphorylation had occurred via a cytosolic tyrosine kinase activated by bpV(phen) seems unlikely since cytosolic tyrosine kinase activities were not augmented either in the absence or presence of colchicine treatment (I). Additionally in hepatoma tissue culture cells overexpressing kinase negative IRK (K1030A mutation) versus those overexpressing normal IRK, treatment with bpV(phen) resulted in no phosphorylation of IRS-1 in the former but IRK activation and IRS-1 phosphorylation in the latter.() Effect of bpV(phen) on Plasma Glucose Levels and 2-[H]DG Uptake in Rat Diaphragm-We have previously shown that bpV(phen) administration alone causes hypoglycemia in rats (19) . Based on the inability of bpV(phen) to stimulate glycogen synthesis in muscle (20) we have suggested that the locus of bpV(phen) action is the liver and not muscle for producing the hypoglycemic response. An hepatic locus of bpV(phen) action was further supported by the observation that bpV(phen) did not stimulate 2-deoxyglucose uptake into rat skeletal muscle in contrast to the significant stimulation induced by insulin (Fig. 6 A). It is of interest that following the selective activation of the endosomal IRK produced by bpV(phen) in colchicine pretreated rats, hypoglycemia was induced (Fig. 6 B) to a similar degree to that achieved with bpV(phen) in the absence of colchicine pretreatment (19) .


Figure 5: Phosphotyrosine content of IRS-1 following bpV(phen) treatment in the presence and absence of colchicine. Rats were fasted overnight and given an intrajugular injection of 0.6 µmol/100 g body weight bpV(phen). In some cases rats were injected intrajugularly with 25 µmol/100 g body weight colchicine in 0.9% saline 1 h prior to bpV(phen) injection. A 20% liver homogenate was prepared as described under ``Experimental Procedures'' and the cytosolic fraction separated following centrifugation at 200,000 g for 45 min. IRS-1 was immunoprecipitated from the cytosol by IRS-1, the pellet washed three times, boiled in Lammeli sample buffer, and subjected to SDS-PAGE (7.5% gel) as described under ``Experimental Procedures.'' PY and IRS-1 content of the immunoprecipitates were determined by Western blotting using specific antibodies, visualized by autoradiography, and quantified by laser densitometry. Panel A, immunoblotting of immunoprecipitated IRS-1 with antibodies to PY and IRS-1, respectively. Panel B, the quantification of PY and IRS-1 immunoblots following laser densitometry of the autoradiograph. *, p < 0.02; **, p < 0.01.




Figure 6: Effect of bpV(phen) on 2-deoxyglucose uptake into rat skeletal muscles and blood glucose levels in colchicine pretreated rats. Panel A, uptake of 2-[H]DG into diaphragm ( DIA), soleus, tibialis anterior ( TA), and extensor digitorum longus ( EDL) was assessed in overnight fasted rats. Animals were sacrificed 15 min following intrajugular injection of a combination of 18.75 µCi/100 g body weight 2-[H]DG with either PBS, 0.6 µmol/100 g body weight bpV(phen) or 1.5 µg/100 g body weight insulin. Muscles were analyzed for their content of 2-[H]DG-6-phosphate as described under ``Experimental Procedures.'' The content of 2-[H]DG-6-phosphate in rat skeletal muscles: DIA, soleus, TA, and EDL is shown following PBS ( open bars), bpV(phen) ( slanted bars), or insulin ( solid bars) treatment. Each observation is the mean ± S.E. of determinations performed on three to six separate animals. *, p < 0.05. Panel B, rats were fasted for 4 h and then injected with 25 µmol/100 g body weight colchicine in 0.9% saline or 0.9% saline alone via the tail vein. One hour later rats received a second injection of either PBS or bpV(phen) (0.6 µmol/100 g body weight) via the same route. Tail blood samples were collected every 20 min for 3 h into heparinized Eppendorf tubes and centrifuged for 5 min at 14,000 g. Plasma glucose concentration was determined in duplicate using a glucose analyzer-2 (Beckman Instruments). The effect of bpV(phen) following colchicine treatment () on plasma blood glucose levels is expressed as a percent of plasma glucose levels following colchicine treatment alone (). Each point is the mean ± S.E. of determinations performed on 9-10 animals.




DISCUSSION

Earlier studies found that the administration of insulin is rapidly followed by the accumulation of both insulin (33) and IRs (26) in rat liver ENs. It was subsequently shown that these internalized receptors were activated IRKs (7) whose autophosphorylation activity exceeded the maximal levels attained by PM IRKs (7) . These observations on the IRK and comparable studies on the epidermal growth factor receptor kinase (34, 35, 36) support a role for internalized receptors in transmembrane signaling (37) . In this paper we have described for the first time an in vivo model in which there is selective activation of the hepatic endosomal IRK following which there is tyrosine phosphorylation of IRS-1 and hypoglycemia.

The data of Fig. 1 suggest that bpV(phen) acts following entry into the cell. Fig. 2 shows that ENs contain an IRK-associated PTP(s) whose inhibition by bpV(phen) followed by insulin administration leads to pronounced activation of the endosomal IRK. The striking correlation between IRK activation on one hand and PTP(s) inhibition on the other supports our earlier suggestion (19) that these phenomena are causally related. The relationship between PTP inhibition and IRK activation deserves further comment as various studies have observed that cultured cells display a low level of basal IRK activity even in the absence of insulin (9, 19) . It is envisioned that in the basal state a low level futile cycle operates in which phosphotyrosine is formed and degraded with no significant net autophosphorylation. The administration of bpV(phen) disturbs this equilibrium consequent to the inhibition of dephosphorylation leading to net IRK autophosphorylation and hence activation. The time course of IRK activation following bpV(phen) was slower than that following insulin which was maximal at 2 min post-injection (7, 8, 19) . This is consistent with the different modes by which each agent promotes IRK activation. Thus bpV(phen) activates the IRK indirectly by inhibiting IRK-associated PTP(s), whereas insulin activates the IRK by augmenting kinase activity directly.

To assess the primary site of action of bpV(phen) we evaluated the kinetics of PM and EN IRK activation following bpV(phen) administration. As seen in Fig. 3, bpV(phen) administration produced a more rapid and greater activation of the IRK in ENs compared to those of PM. This prompted the suggestion that the lag in and lower level of PM IRK activation reflected a recycling of IRKs primarily activated in ENs. We therefore attempted to abrogate receptor recycling by pretreating the animals with the colchicine, a well known inhibitor of microtubule function. Indeed colchicine treatment completely prevented the increase in PM IRK activity following bpV(phen) administration without affecting the extent of endosomal IRK activation. One hour of colchicine treatment did not activate the IRK in PM or ENs as measured by either the exogenous kinase assay or IRK tyrosine phosphorylation ( cf. zero time in Figs. 3 and 4). Nor did colchicine pretreatment alter the effect of insulin on the extent of IRK activation in either PM or ENs (see ``Results''). Therefore the most likely explanation for the selective loss of bpV(phen)-induced IRK activation in the PM after colchicine administration is the inhibition of exocytic transport of the activated endosomal IRK to the PM.

These findings defined an in vivo system in which endosomal IRK could be selectively activated following colchicine and bpV(phen) treatment, thus allowing us to assess the possibility of signaling from this compartment alone. IRS-1, an important substrate of the IRK, has been implicated in aspects of the insulin signaling pathway (38, 39) . Our study has shown that IRS-1 became highly tyrosine phosphorylated by 5 min following bpV(phen) treatment alone and by 15 and 30 min following colchicine pretreatment, instances when the endosomal but not PM IRK was active. This implies that the endosomal IRK was capable of normal tyrosine phosphorylation of IRS-1 and hence of signaling. The level of IRS-1 tyrosine phosphorylation was comparable to that seen with in vivo administered insulin (1.5 µg/100 g body weight).() It was further seen that bpV(phen) treatment caused recruitment of IRS-1 to the cytosol. This recruitment to the cytosol suggests that in the nonphosphorylated state IRS-1 may be associated with cell membrane fractions, including ENs (40) , possibly via direct interactions with the IRK (41) . The recruitment to cytosol may be a necessary part of the IRK signaling pathway allowing docking with other signal transducers and facilitating their interactions with downstream elements in the signaling pathway (38, 39) . However, it cannot be excluded that tyrosine phosphorylation of IRS-1 renders it more immunoreactive which could augment both the amount of IRS-1 immunoprecipitated and/or the strength of the signal generated on immunoblotting.

Earlier work has suggested that muscle accounts for 50% (24) , liver for 30% (42) , and adipose tissue for <10% (24) of the uptake of administered glucose. Several observations have indicated that the in vivo hypoglycemic action of bpV(phen) derives primarily from action on the liver rather than on skeletal muscle. Thus bpV(phen) could not stimulate the incorporation of glucose into glycogen (20) or the uptake of 2-deoxyglucose (Fig. 6 A) into skeletal muscles. Thus our observation that bpV(phen) administration would still lower blood glucose levels in colchicine-pretreated rats (Fig. 6 B) implicates the endosomal IRK of rat liver in effecting reduced hepatic glucose output.

The hypothesis that internalization of receptors may be involved in transmembrane signaling (37, 43) has been supported by observations on the accumulation of activated IRKs (7, 8) and epidermal growth factor receptor kinases (34, 36) in ENs. This hypothesis has been strengthened by the recent demonstration that accumulation of activated epidermal growth factor receptor kinases in ENs leads to the phosphorylation of an in vivo substrate, pyp55 (44) (also called p55 (45) and more recently identified as SHC (46, 47) ), leading to the association of SHC and GRB2 with the activated internalized epidermal growth factor receptor kinase (46) . The current study has shown that selective activation of the hepatic endosomal IRK leads to phosphorylation and recruitment of IRS-1 to the cytosol and the lowering of blood glucose levels. It thus provides support for a role for the EN in insulin signal transduction and illustrates the importance of the intracellular IRK for the realization of some of the metabolic effects of insulin.

  
Table: Immunoprecipitation of lectin-purified PM and EN receptors with 960

Rats were fasted overnight and sacrificed 15 min after the injection of 0.6 µmol/100 g body weight of bpV(phen). Hepatic PM and ENs were prepared, solubilized, and IRs partially purified by lectin chromatography as described under ``Experimental Procedures.'' Immunoprecipitations were performed in parallel with 960 or control IgG on these partially purified IRs. Subsequent supernatants were assayed for tyrosine kinase activity (TKA) and I-insulin binding (B) as described under ``Experimental Procedures.'' The percentage of tyrosine kinase activity and B immunoprecipitated was determined by subtracting supernatant values for the immunoprecipitations with 960 from those derived with control IgG. All values are the mean ± S.E. of determinations on three to five separate animals.


  
Table: Effect of bpV(phen) and colchicine on IR content of PM and ENs

Rats were fasted overnight and injected intrajugularly with 0.6 µmol/100 g body weight bpV(phen) 60 min after receiving an intrajugular injection of colchicine (25 µmol/100 g body weight) or vehicle. At the specified time animals were sacrificed and PM and ENs prepared. I-Insulin binding was determined as described under ``Experimental Procedures.'' All values are the mean ± S.E. from the noted number (parentheses) of separate animals.


  
Table: Effect of bpV(phen) and colchicine on cytosolic tyrosine kinase activities

Rats were fasted overnight and injected intrajugularly with 0.6 µmol/100 g body weight bpV(phen) with or without colchicine pretreatment (25 µmol/100 g body weight). At the specified time animals were sacrificed and cytosol prepared. Tyrosine kinase activity (TKA) was assayed as described under ``Experimental Procedures.'' All data are the mean ± S.E. from the noted number ( n) of separate animals.



FOOTNOTES

*
This work was supported by grants from the Medical Research Council of Canada, Nordic Merrell Dow Research, Laval, PQ, Canada, and the Maurice Pollack Foundation, Montreal, PQ, Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Previously supported by a fellowship from the Royal Victoria Hospital Research Institute and currently the recipient of a fellowship from the Juvenile Diabetes Foundation International.

Prior recipient of a fellowship from the Juvenile Diabetes Foundation International.

**
Recipient of a fellowship from the Royal Victoria Hospital Research Institute.

§§
To whom correspondence should be addressed: Polypeptide Hormone Lab., Strathcona Anatomy & Dentistry Building, 3640 University St., Rm. W3.15, Montreal, Quebec, H3A 2B2, Canada. Tel.: 514-398-4101; Fax: 514-398-3923; E-mail: mc85@musica.mcgill.ca.

The abbreviations used are: IR, insulin receptor; bpV(phen), bisperoxo(1,10-phenanthroline)oxovanadate(v) anion; BSA, bovine serum albumin; 2-[H]DG, 2-deoxy[1-H]glucose; EN, endosome; IRK, insulin receptor tyrosine kinase; IRS-1, insulin receptor substrate-1; PGT, Glu:Tyr, polyglutamic acid-tyrosine (4:1); PM, plasma membrane; PMSF, phenylmethanesulfonic fluoride; PTP, phosphotyrosine phosphatase; WGA, wheat germ agglutinin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PY, -phosphotyrosine; PAGE, polyacrylamide gel electrophoresis.

All studies herein cited were performed with the approval of the McGill University Animal Care Committee.

A. Dorato, J. J. M. Bergeron, and B. I. Posner, manuscript in preparation.

J.-O. Contreres, V. Dumas, A. Shaver, and B. I. Posner, manuscript in preparation.

A. P. Bevan, P. G. Drake, A. Shaver, J. J. M. Bergeron, and B. I. Posner, manuscript in preparation.


ACKNOWLEDGEMENTS

We express our gratitude to Dr. Morris White for generously providing the antibodies to IRS-1 used in these studies. We also thank Gerry Baquiran and Qingwei Chu for their excellent technical assistance and Dr. Jesse B. Ng for preparing the pV compound used in these studies.

Addendum-Subsequent to the submission of this manuscript, Kublaoui et al.(48) demonstrated that in rat adipocytes IRS-1 was distributed between internal membranes (20%) and cytosol (80%) with none detectable in the plasma membrane. Following insulin treatment, tyrosine phosphorylation of IRS-1 paralleled that of the IRK in internal membranes whereas no tyrosine-phosphorylated IRS-1 was observed in plasma membranes. This observation is consistent with insulin signal transduction occurring within the endosomal compartment.


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