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Endocrinology Vol. 141, No. 11 4041-4049
Copyright © 2000 by The Endocrine Society

ARTICLES

Compartmentalization and Insulin-Induced Translocations of Insulin Receptor Substrates, Phosphatidylinositol 3-Kinase, and Protein Kinase B in Rat Liver1

Alejandro Balbis, Gerry Baquiran, John J. M. Bergeron and Barry I. Posner

Polypeptide Hormone Laboratory and Department of Anatomy and Cell Biology (J.J.M.B.), McGill University, Montréal, Québec, Canada H3A 2B2

Address all correspondence and requests for reprints to: Dr. B. I. Posner, Polypeptide Hormone Laboratory, Strathcona Anatomy and Dentistry Building, 3640 University Street, Room W3.15, Montréal, Québec, Canada H3A 2B2. E-mail: mc85{at}musica.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling proteins between subcellular compartments in rat liver. In plasma membranes (PM) and microsomes, insulin induced a rapid decrease in insulin receptor substrate-1/2 (IRS1/2) within 30 sec and an increase in these proteins in endosomes (EN) and cytosol. The level of p85 in PM increased 2.3-fold at 30 sec after insulin stimulation followed by a decrease at 2 min. In this interval, 60–85% and 10–20% of p85 in PM was associated with IRS1 and IRS2, respectively. Thus, in PM, IRS1/2 accounts for almost all of the protein involved in phosphatidylinositol 3-kinase activation. In ENs insulin induced a maximal increase of 40% in p85 recruitment. As in PM, almost all p85 was associated with IRS1/2. The greater level of p85 recruitment to PM was associated with a higher level of insulin-induced recruitment of Akt1 to this compartment (4.0-fold in PM vs. 2.4-fold in EN). There was a close correlation between Akt1 activity and Akt1 phosphorylation at Thr308 and Ser473 in PM and cytosol. However, in ENs the level of Akt1 activity per unit of phosphorylated Akt1 was significantly greater than in PM, indicating that in addition to phosphorylation, another factor(s) modulates Akt1 activation by insulin in rat liver. Our results demonstrate that activation of the insulin receptor kinase and modulation of key components of the insulin signaling cascade occur at the cell surface and within the endosomal system. These data provide further support for the role of the endocytic process in cell signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AFTER THE BINDING of insulin to its surface receptor, the insulin receptor kinase (IRK) undergoes tyrosine phosphorylation of its ß-subunit and kinase activation. The activated IRK is essential for realizing insulin action. This involves the phosphorylation of substrate adaptor proteins, principally insulin receptor substrate-1 (IRS1) and IRS2, on tyrosine residues that interact with SH2 domain-containing proteins to generate downstream signals (1, 2). Phosphatidylinositol 3-kinase (PI3-kinase), a protein with such a domain in its regulatory subunit (p85), is activated by interaction with IRS proteins (3). PI3-kinase phosphorylates inositol phospholipids at the 3-hydroxy position of the inositol ring, resulting in the generation of products essential for insulin metabolic actions such as GLUT4 translocation, glycogen synthase activation, and insulin-induced mitogenesis (3).

After insulin binding, the activated IRK is rapidly internalized into endosomes (ENs) (4, 5). In this acidic compartment, insulin is released from the IRK and degraded (6, 7), whereas the latter recycles to the cell surface or undergoes degradation (8, 9). With time there have been increasing data indicating that endocytosis of activated IRK plays a role in insulin transmembrane signaling (5, 10, 11, 12) and in the signaling mechanisms of other ligand-receptor complexes (13, 14).

The above suggests that intracellular compartments are important in the generation of downstream signaling events. Other data implicate compartmentalization of downstream signaling molecules as a significant component of the signaling process. Thus, insulin treatment of 3T3-L adipocytes promoted the association of IRS1 with PI3-kinase in internal membranes, and translocation of IRS1 from this compartment to cytosol (15, 16, 17). Furthermore, it has been suggested that the specific intracellular location of different activated signaling molecules is a determinant of signaling specificity. For example, although both platelet-derived growth factor (PDGF) and insulin stimulate PI3-kinase activity in adipocytes, only insulin promotes translocation of the glucose transporter (GLUT4) from intracellular vesicles to the cell surface (18). PDGF principally recruits PI3-kinase to plasma membrane (PM), whereas insulin induces association and translocation of this kinase mainly to intracellular compartments (19, 20).

Protein kinase B, also known as Akt/PKB, has been implicated as a downstream protein kinase mediating insulin responses, including insulin-induced glucose uptake and glycogen synthase activation (21). The activation of Akt is associated with its translocation from cytosol to PM. The mechanism appears to involve binding via its pleckstrin homology domain to products of PI3-kinase activation [i.e. PI(3, 4)P2 and PI(3, 4, 5)P3] generated at the PM (22, 23), accompanied by phosphorylation at Thr308 and Ser473 (24). The data on cellular translocation of Akt1 are largely based on morphological analyses, and little has been done to document this in well defined cell fractions.

The above observations indicate the importance of compartmentalization in insulin signaling and point to the need to delineate this process in more detail. In the present study we sought to evaluate the subcellular distribution of key insulin-signaling molecules in well defined subcellular fractions from rat liver. Three compartments were examined: PM, ENs, and cytosol. After insulin treatment, IRS1 was observed to be the main docking protein for PI3-kinase. Insulin induced the translocation of Akt1 to membranes and its activation in all compartments within 30 sec. We found a lack of correlation between Akt1 phosphorylation at Thr308 and Ser473, and Akt1 kinase activity, suggesting that another factor(s), in addition to these site-specific phosphorylations, regulates Akt1 activation. Our results demonstrate that insulin-induced translocation of key signaling proteins is dynamic and rapid in all of the compartments analyzed, and that ENs represent an important compartment for the recruitment and activation of insulin-signaling proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female Sprague Dawley rats, 10 weeks of age (160–180 g BW) were purchased from Charles River Laboratories, Inc., Canada Ltd. (St. Constant, Canada), housed in an animal facility with 12-h light cycles at 25 C, and fed Purina chow (Ralston Purina Co., St. Louis, MO) ad libitum. Animals were fasted overnight (16–18 h) before use.

Materials
Porcine insulin was a gift from Eli Lilly & Co. (Indianapolis, IN). Phenylmethylsulfonylfluoride (PMSF), HEPES (free acid), sodium orthovanadate, Tris, and most other chemicals were purchased from Sigma (St. Louis, MO). Protein A-Sepharose CL-4B (PAS) was obtained from Pharmacia Biotech (Uppsala, Sweden). Protein G-agarose and ATP (disodium salt) were obtained from Roche Molecular Biochemicals (Laval, Canada). Microcystin-LR was purchased from Calbiochem (La Jolla, CA). [{gamma}-32P]ATP (3000 Ci/mmol), [125I]goat antimouse and [125I]goat antirabbit secondary antibodies were purchased from NEN Life Science Products (Lachine, Canada). Reagents for electrophoresis were obtained from Bio-Rad Laboratories, Inc. (Richmond, CA). Kodak X-OMAT AR film was purchased from Picker International (Montreal, Canada). Polyvinylidene difluoride Immobilon-P transfer membranes were obtained from Millipore Corp. (Mississauga, Canada). An antibody raised against a peptide corresponding to residues 942–969 of the juxtamembrane region of the IRK ß-subunit (anti-960) was prepared and purified on a PAS column as previously described (25). Polyclonal anti-p85 and anti-IRS2 for immunoprecipitation and Western blotting, a polyclonal anti-IRS1 for Western blotting, specific anti-Akt1, and a substrate peptide for the Akt1 kinase assay (Crosstide) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonals antiphospho-Akt (Thr308 and Ser473) for Western blotting were purchased from New England Biolabs, Inc. (Beverly, MA). A monoclonal antiphosphotyrosine antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A polyclonal antibody raised against a peptide corresponding to the 14 carboxyl-terminal residues of IRS1 was used for immunoprecipitation.

Preparation of subcellular fractions
Rats were anesthetized and killed by decapitation at the indicated times after intrajugular injections as described in the text. Livers were exsanguinated, rapidly excised, and minced at scissor point in ice-cold buffer (5 mM Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose, 1 mM benzamidine, 1 mM PMSF, 1 mM MgCl2, 2 mM NaF, and 2 mM sodium orthovanadate). PM, ENs, and cytosol were prepared as previously described (5). The protein content of these fractions was measured using a modification of Bradford’s method (26) with BSA as standard.

Immunoprecipitation and immunoblotting
Cell fractions (500 µg protein) in a final volume of 550 µl were incubated in the presence of 1% (vol/vol) Triton X-100 and 0.5% (wt/vol) sodium deoxycholate at 4 C for 1 h. Supernatants were incubated for 2 h with either anti-IRS1 or anti-IRS2 at 4 C, after which 50 µl of a 50% slurry of PAS was added, and the solution was incubated for an additional 1 h. After centrifugation the pellet was washed three times with 1 ml wash buffer (50 mM HEPES, pH 7.4, containing 1% Triton X-100, 150 mM NaCl, 100 mM NaF, and 2 mM Na3Vo4) followed by boiling in Laemmli sample buffer. Samples (immunoprecipitates or intact samples) were subjected to SDS-PAGE (7.5% gel) and then transferred to Immobilon-P membranes for Western blotting. Either [125I]goat antirabbit or [125I]goat antimouse antibodies were used as the secondary antibody, and after autoradiography at -80 C, appropriate bands were quantified using a GS-700 Imaging Densitometer (Bio-Rad Laboratories, Inc.)

Akt1 kinase activity
Cell fractions (500 µg) were incubated in buffer A [50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3Vo4, 0.1% (vol/vol) 2-mercaptoethanol, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium {alpha}-glycerophosphate, 0.1 mM PMSF, and 1 µM Microcystin-LR] at 4 C for 1 h. After centrifugation, the supernatant was incubated with protein G-Sepharose-Akt1 antibody complex for 2 h at 4 C. The Akt1 immunoprecipitate was washed twice with 500 µl buffer A containing 0.5 M NaCl, twice with 500 µl buffer B [50 mM Tris-HCl (pH 7.5), 0.03% (wt/vol) Brij 35, 0.1 mM EGTA, and 0.1% (vol/vol) 2-mercaptoethanol], and once with 500 µl ADB [20 mM MOPS (pH 7.2), 25 mM {alpha}-glycerol phosphate, 1 mM Na3Vo4, and 1 mM dithiothreitol]. In vitro kinase assays were performed for 10 min at 30 C in 60 µl ADB containing 10 µM PKA inhibitor peptide, 30 µM Crosstide, 10 mM MgCl2, and 25 µM [{gamma}-32P]ATP (10 µCi). The assays were terminated and analyzed as previously described (27).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of insulin on subcellular distribution of IRK and IRS1/2 in rat liver
As previously observed (25) in vivo insulin administration (1.5 µg/100 g BW) effected a rapid decrease in the number of PM IRKs, an increase in the phosphotyrosine content of PM IRKs, and the accumulation within ENs of tyrosine-phosphorylated IRKs (Fig. 1Go). IRK phosphotyrosine content reached a peak in PM at 30 sec and in ENs at 2 min postinjection, with a subsequent rapid decrease in both fractions (Fig. 1Go).



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  		Figure 1. Time course of insulin-stimulated IRK ß-subunit tyrosine phosphorylation in PM and EN. Rats were fasted overnight, injected with insulin (1.5 µg/100 g BW), and killed at the indicated times thereafter. PM and ENs were purified, and samples (50 µg protein) were subjected to SDS-PAGE (7.5%) and transferred to Immobilon-P membranes as described in Materials and Methods. Phosphotyrosine and IRK ß-subunit contents were assessed by immunoblotting using antiphosphotyrosine and anti-960 antibodies, respectively.

 
This rapid phosphorylation correlates with activation of the IRK and tyrosine phosphorylation of its substrates, of which IRS1 and IRS2 are the best characterized. We thus examined the influence of insulin administration on the association of these proteins with ENs, PM, and cytosol. As shown in Fig. 2Go (left panels) insulin administration induced a rapid and significant increase in the content of IRS1 in cytosol (60% over control at 30 sec and 2 min; P < 0.01) and in ENs (111% over control at 2 min; P < 0.05), whereas the PM level of IRS1 decreased by 44% and 28% (P < 0.01 vs. controls) at 2 and 5 min, respectively.



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  		Figure 2. Subcellular distribution of IRS1 and IRS2 in rat liver. Rats were fasted overnight, then received a single iv dose of insulin (1.5 µg/100 g BW), and were killed at the noted times thereafter. PM ({circ}), EN (•), and cytosol ({blacktriangleup}) were prepared as described in Materials and Methods. Aliquots (50 µg protein) were subjected to SDS-PAGE (7.5%) and analyzed by immunoblotting with anti-IRS1 or anti-IRS2 antibodies. Upper panel, representative immunoblot. Lower panel, levels of IRS1/2 in each fraction were quantitated using scanning densitometry, and the results plotted as a percentage of the value in noninsulin-treated (control) animals as described in Materials and Methods. Each point is the mean ± SE of four independent experiments. *, P < 0.05; #, P < 0.01 (vs. control, by Student’s t test).

 
Similar results were obtained when IRS2 was examined. After insulin administration, the IRS2 concentration in cytosol increased significantly by 46% (P < 0.01 vs. control) and 44% (P < 0.05 vs. control) at 2 and 5 min, respectively, whereas IRS2 recruited to ENs was 2.4-fold higher than the basal level between 2 and 5 min (Fig. 2Go). IRS2 content in PM showed a significant decrease at 2 min (52%; P < 0.01 vs. control) and 5 min (41%; P < 0.01 vs. control) after insulin injection (Fig. 2Go). Both IRS1 and IRS2 returned to near-basal values in all of the subcellular fractions studied by 15–30 min after insulin administration.

The time course of IRK content in ENs and PM is mirrored by those of IRS1 and IRS2 in these compartments. We therefore sought to determine whether IRKs were internalized as a complex with IRS1 and/or IRS2. To this end we looked for the presence of these substrates in IRK immunoprecipitates from PM and ENs. We detected a modest association between IRS1 and IRK in PM, but the level of IRS1 in IRK immunoprecipitates was very low compared with the total IRS1 content of this compartment (data not shown). No association between IRS2 and IRK was found in any compartment. These observations do not exclude the existence of weak associations of the IRSs with the IRK that do not survive the solubilization and immunoprecipitation procedures.

Effect of insulin on the subcellular distribution of p85 in rat liver
We subsequently analyzed the subcellular distribution of p85 after insulin stimulation. Insulin caused a 2.3-fold increase (P < 0.01 vs. control) in the PM content of p85 at 30 sec, followed by a rapid decrease to steady state by 5 min postinjection (Fig. 3aGo). The level of p85 in ENs increased 30% at 30 sec and did not change significantly over the next 30 min. The distribution of p85 in cytosol showed no significant change throughout the 30-min period after insulin injection (Fig. 3aGo).



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  		Figure 3. Subcellular distribution of p85 in rat liver. PM ({circ}), ENs (•), and cytosol ({blacktriangleup}) were prepared, subjected to SDS-PAGE (7.5%), and transferred to Immobilon-P as described in Fig. 2Go. Membranes were analyzed by immunoblotting with a specific anti-p85 antibody as described in Materials and Methods. The results are plotted as a percentage of the value in nonstimulated (control) rats. a, Representative immunoblot (top) and densitometric analysis (bottom). b, Cell fractions (500 µg protein) were immunoprecipitated with anti-IRS1 or anti-IRS2 antibodies, and the immunoprecipitates were subjected to SDS-PAGE (7.5%) and transferred to Immobilon-P membranes followed by immunoblotting with anti-p85 antibody. Each point is the mean ± SE of four to six independent experiments. *, P < 0.05; #, P < 0.01 (vs. control, by Student’s t test).

 
We investigated the insulin-induced association of IRS1 and IRS2 with p85 in ENs, PM, and cytosol. Equal amounts of subcellular fraction protein were subjected to immunoprecipitation with IRS1 or IRS2 and were subsequently immunoblotted with an anti-p85 antibody. In these studies a clear association of p85 with the IRSs could be documented. In PM, p85 association with IRS1 and IRS2 was maximum at 30 sec and declined rapidly thereafter (Fig. 3bGo). Insulin stimulation also increased the association of IRS1 and IRS2 with p85 in cytosol and ENs as early as 30 sec (Fig. 3bGo).

We next sought to determine the proportion of p85 binding to IRS1 and IRS2, respectively. This should define the extent to which each IRS contributed to insulin-induced PI3-kinase activation, as it has been observed that in rat liver there is a good correlation between PI3-kinase activity and the amount of p85 in IRS1 immunoprecipitates (28). From Fig. 3bGo alone one cannot deduce the extent of binding of p85 to IRSs, because the efficiency of immunoprecipitation and immunoblotting depends on the antibody used and perhaps on the subcellular fraction being analyzed. We estimated the relative extent of p85 association with a particular IRS by comparing the ratio of p85/IRS in intact subcellular fractions with that ratio in IRS immunoprecipitates. One can view the ratio of p85/IRS in intact samples as a measure of the maximum possible association if all of the p85 were associated with only the particular IRS being evaluated. Thus, when the ratio of p85/IRS in the IRS immunoprecipitates was expressed as a percentage of p85/IRS in the intact samples, we obtained a measure of the association of p85 with the IRS that excluded the impact of immunoprecipitation efficiency. By using this method we found that in the basal state p85 scarcely binds to IRS1or2, but after insulin stimulation the majority of p85 is associated with IRS1 (60–85%) and IRS2 (10–20%) in both PM and ENs (Table 1Go). In a previous work we showed association of p85 with IRK in PM (28A ) and demonstrated that 5–10% p85 was bound to IRK after insulin stimulation (data not shown). As approximately 90% of p85 is bound to IRS proteins, we concluded that in PM, after insulin stimulation, almost all p85 is associated with IRS1/2 and IRK. In cytosol, approximately 40% and 25% of p85 are associated with IRS1 and IRS2, respectively, after insulin stimulation (Table 1Go). Thus, although a large majority of p85 is associated with IRS1 and IRS2 in PM and ENs, approximately 30–40% of p85 in cytosol is free or associated with other proteins. The data further indicate that in the membrane fractions (PM and EN), IRS1 is the predominant protein involved in PI3-kinase activation.


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  		Table 1. Percentage of p85 bound to IRS1 and IRS2 in EN, PM, and cytosol

 
Insulin-induced localization of Akt1 in subcellular compartments
Akt/PKB is an important target of PI3-kinase signaling. As shown above, PI3-kinase activation by insulin plays a role in all subcellular compartments studied. As Akt is a downstream protein kinase that functions as an important mediator of IRK activation, we studied its distribution and activation in PM, ENs, and cytosol. It was previously shown in hepatocytes that insulin stimulated Akt1/PKB{alpha} activity 4-fold over that of Akt2/PKBß and had no effect on Akt3/PKB{gamma} (29). Consequently, we focused our studies in intact liver on the recruitment and activation of Akt1. As in the above studies of p85 and IRSs, hepatocyte subcellular fractions were prepared after the in vivo injection of insulin (1.5 µg/100 g BW), and the content of Akt1 was determined by immunoblotting. As shown in Fig. 4Go, the content of Akt1 in PM increased by about 400% at 30 sec to 2 min after insulin stimulation (P < 0.001 vs. control). Thereafter, the level declined almost to basal by 15 min. In ENs, insulin-induced recruitment of Akt1 increased by 30 sec and reached a steady state level by 5 min, which was 2.4-fold the basal level. No change in the distribution of Akt1 was observed in cytosol after insulin stimulation (Fig. 4Go, top panel). The above data indicate that the time courses of Akt1 translocation and p85 recruitment to PM and ENs were similar.



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  		Figure 4. Subcellular distribution of Akt1 in insulin-stimulated rat liver. PM ({circ}), ENs (•), and cytosol ({blacktriangleup}) were prepared and subjected to SDS-PAGE (7.5%) as described in Fig. 2Go. Samples were transferred to Immobilon-P membranes and immunoblotted with a specific antibody against Akt1/PKB{alpha}. Upper panel, Representative immunoblot with anti-Akt1. Lower panel, Densitometric quantitation of Akt1 levels. The results are expressed as a percentage of the Akt1 levels in PM at 30 sec. Each point represents the mean ± SE of four to six independent experiments. *, P < 0.05; #, P < 0.001 (vs. control, by Student’s t test).

 
This correlation was further studied subsequent to the administration of bpv(phen), a potent inhibitor of phospho-Tyr phosphatases, that maximally increased the phosphotyrosine content and exogenous tyrosine kinase activity of the IRK in PM and ENs by 15 min (12). We found that bpv(phen) induced a parallel increase in the contents of both p85 and Akt1 in PM, with maximum stimulation (3-fold increase over basal) at 15 min postinjection (data not shown). Taken together these data support the proposed model that the products of PI3-kinase are involved in directing the translocation of Akt to PM.

Figure 4Go (top panel) also shows that insulin induced a shift in the electrophoretic mobility of Akt1 in PM. This was less evident in ENs and cytosol, suggesting that Akt1 in PM may be more highly phosphorylated. We investigated the state of Akt1 phosphorylation by immunoblotting with specific antibodies against Akt-phospho-Thr308 and Akt-phospho-Ser473. The results in Fig. 5Go (top panel) show that Akt1 is phosphorylated on both Ser473 and Thr308 within 30 sec of insulin administration, with a gradual decrease after 5 min in the three subcellular compartments studied. The level of Akt1 phosphorylation was evaluated by comparing the ratio of phospho-Akt/Akt1 within each compartment. Figure 5Go (lower panel) shows, as previously suggested in Fig. 4Go, that after insulin stimulation the proportion of Akt1 phosphorylated on Ser473 and Thr308 is higher in PM than in ENs and cytosol. By 30 min after insulin administration Akt1 is almost fully dephosphorylated in all three subcellular compartments studied (Fig. 5Go, lower panel). These data indicate that physiological concentrations of insulin in vivo induce the rapid (within 30 sec) recruitment and phosphorylation of Akt1 in both PM and ENs.



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  		Figure 5. Insulin-stimulated phosphorylation of Akt1 in rat liver subcellular fractions. PM ({circ}), ENs (•), and cytosol ({blacktriangleup}) were prepared as described in Materials and Methods. Cell fractions (50 µg protein) were subjected to SDS-PAGE and immunoblot analysis with specific antibodies against Akt1, Akt-Ser473, or Akt-Thr308. Upper panel, Representative Western blot showing Akt1 phosphorylated at Ser473 and Thr308. Lower panel, Levels of both Akt1 and site-specific phosphorylated Akt were quantitated by scanning densitometry. The results are expressed as a percentage of the ratio of phosphorylated Akt/Akt1 in PM at 30 sec. Each point is the mean ± SE of three independent experiments.

 
Insulin-induced activation of Akt1 in subcellular compartments
Next we determined the effect of insulin administration on the level of Akt1 kinase activity. Equal quantities of protein from ENs, PM, and cytosol were subjected to immunoprecipitation with Akt1 antibodies and then incubated with Crosstide, a substrate for the Akt1 kinase assay. Insulin-induced Akt1 activity was maximum between 30 sec and 2 min in all compartments studied (Fig. 6aGo). Akt1 kinase activity increased approximately 20-fold in PM and cytosol after insulin stimulation. In ENs the activity increased from a nonmeasurable basal level to one about one seventh that observed in PM (Fig. 6aGo). Akt1 activity, immunoprecipitated from insulin-stimulated fractions by control IgG, did not differ from background, indicating that all kinase activity was probably associated with Akt1. Although the kinase activity remained elevated 15 min after insulin stimulation in ENs and cytosol, during the same period of time the activity returned to near-control values in PM (Fig. 6aGo).



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  		Figure 6. Time course of insulin-stimulated Akt1 activity in rat liver cell fractions. PM ({circ}), ENs (•), and cytosol ({blacktriangleup}) were prepared and solubilized, and Akt1 was immunoprecipitated from aliquots (500 µg protein) using a specific anti-Akt1 antibody as described in Materials and Methods. Immunoprecipitates were assayed for kinase activity with Crosstide as substrate or were subjected to SDS-PAGE, transferred to Immobilon-P membranes, and immunoblotted with an anti-Akt1 antibody. a, Representative experiment showing Akt1 activity (arbitrary units) after insulin stimulation. b, Ratio of Akt1 activity/Akt1 expressed as a percentage of PM at 30 sec. Each point is the mean ± SE of three independent experiments.

 
We then investigated the relationship between Akt1 activity and its level of phosphorylation by studying the correlation between Akt1 phosphorylation and Akt1 activitation. The ratio of Akt1 activity/Akt1 calculated in Akt1 immunoprecipitates was 2-fold higher in PM than that in cytosol (Fig. 6bGo). This is in accordance with the higher ratio of Akt1 phophorylation/Akt1 in intact PM (Fig. 5Go, lower panel). Surprisingly, although the ratio of Akt1 phosphorylation/Akt1 is higher in PM than ENs (Fig. 5Go, lower panel), the ratio of Akt1 activity/Akt1 in ENs was not different from that in PM (Fig. 6bGo). These results raised the possibility that Akt1 was dephosphorylated during the immunoprecipitation procedure. This seems unlikely, because Akt1 in PM immunoprecipitates showed the same shift in the electrophoretic mobility as Akt1 in intact PM samples (data not shown), and the ratio of Akt1 phosphorylation/Akt1 in the immunoprecipitates was similar to that in intact samples (compare Fig. 5Go, lower panel, with Fig. 7aGo). Akt1 activity and Akt1-Ser473 phosphorylation showed a good correlation in PM and cytosol (Fig. 7bGo, and compare Fig. 6bGo with Fig. 5Go, lower panel). The ratio of Akt1 activity/Akt1 phosphorylation in ENs was significantly higher than that in PM (5.5- and 4.2-fold for Akt1 activity/Akt-Ser473 and Akt1 activity/Akt-Thr308, respectively; Fig. 7bGo). This lack of correlation between phosphorylation and activity in ENs and PM suggests that another factor(s) besides phosphorylation at Thr308 and Ser473 contributes to insulin-stimulated Akt1 activity.



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  		Figure 7. Correlation between Akt1 activity and Akt1 phosphorylation in rat liver subcellular fractions. Rats were fasted overnight, received a single iv dose of insulin (1.5 µg/100 g BW), and were killed 2 min thereafter. Rat liver subcellular fractions were prepared as described in Materials and Methods, solubilized, and immunoprecipitated (aliquots of 1 mg protein) using a specific anti-Akt1 antibody. Immunoprecipitates were resuspended and divided into equal aliquots which were used to measure the levels of Akt1 activity, Akt phosphorylation (Thr308 and Ser473), and Akt1 content. a, Ratio of phosphorylated Akt1/Akt1 as a percentage of that in PM. b, Ratio of Akt activity/phosphorylated Akt as a percentage of that in PM. Each bar represents the mean of two animals ± half the difference.

 
Recently, it was reported that Akt interacts with protein kinase C-{zeta} (PKC-{zeta}) in vivo and that this interaction results in a reduction of Akt kinase activity (30). As this association could be related to the regulation of Akt kinase in our study system, we looked for the presence of PKC-{zeta} in Akt1 immunoprecipitates. Although PKC-{zeta} was present in intact samples, no association of this protein with Akt1 was detected in immunoprecipitates (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The binding of insulin to its receptor is rapidly followed by the internalization and concentration of activated insulin-IRK complexes into ENs (5, 10, 25). There is increasing evidence that insulin signaling occurs within this compartment (12, 31, 32, 33). Studies of the internalization of epidermal growth factor (EGF)-EGF receptor complexes indicate prolonged EGF signaling within ENs (13), which may be necessary for full activation of mitogen-activated protein kinase (34). Given the growing appreciation that compartmentalization plays a significant role in cell signaling, we sought in the present study to examine the kinetics of compartmentalization of key insulin-signaling molecules. Concordant with previous reports (25), insulin induced a rapid decrease in PM and a corresponding increase in endosomal activated IRK. These changes were paralleled by changes in the levels of IRS1 and IRS2 in these compartments, consistent with the possibility that IRK was internalized in association with these substrates. The low recovery of IRS1 and IRS2 from IRK immunoprecipitates (data not shown) argues against this, although a weak association, which dissipates during subcellular fractionation and immunoprecipitation, cannot be excluded.

The compartmentalization of IRS1 has been examined in 3T3-L1 adipocytes, where virtually no IRS1 and IRS2 were in PM, and substantial quantities were found in internal membranes (IM) and cytosol (17, 31). Similar results were obtained in adipocytes when low density membranes (LDM) were studied (16, 35). After insulin administration there was phosphorylation of the IRS1 in IM/LDM, followed by its dissociation into cytosol (16, 17, 35). This sequence of events essentially excluded interactions at the PM, and hence differs significantly from our observations in intact liver. This may reflect the disposition of molecules in cultured cells vs. in vivo or tissue-specific differences in compartmentalization. One cannot make a direct comparison between our observations on ENs and those on IM/LDM, because the latter is a more complex fraction, more equivalent to the microsome fraction of rat liver from which ENs are prepared (5). Indeed, we found that in microsomes the content of IRS1/2 was decreased about 50% by 2–5 min after insulin stimulation (data not shown). Based on these considerations we suggest that our EN preparation is more enriched in early ENs compared with the internal membrane fractions isolated from adipocytes. Thus, all of these researchers agree that the levels of IRS1/2 increase in cytosol and decrease in membrane-bound pool(s) after insulin stimulation. However, our work in liver identifies the PM as the important membrane-bound pool involved.

After insulin administration the regulatory subunit of PI3-kinase rapidly increased in PM (within 30 sec) and then rapidly decreased. This may be the result of p85 translocation from cytosol to PM, its interaction with tyrosine-phosphorylated IRS proteins, and the concomitant decrease in these complexes in PM. This hypothesis was supported by our demonstration that after insulin stimulation, the large bulk of p85 is bound to IRS proteins between 30 sec and 2 min, thus explaining the rapid decrease in p85 in intact PM by 2 min. Interestingly, we also found that from 30 sec to 2 min after insulin stimulation, most p85 (60–85%) present in PM was bound to IRS1, and only about 15% was bound to IRS2. As in rat liver PI3 kinase activity correlates well with the level of p85 detectable in anti-IRS1 immunoprecipitates (28), our results suggest that in hepatic PM, IRS1 is the main protein involved in PI3-kinase activation.

Insulin induced a rapid increase in the levels of p85 in ENs, which was associated with IRS1 and IRS2. As in PM, the majority of endosomal p85 associated with IRS1/2, with IRS1 accounting for the bulk of the recruitment. Thus, in addition to insulin-induced concentration of activated IRKs in ENs, there was concentration in this compartment of key signaling molecules involved in propagation of the insulin signal. The kinetics and levels of association are consistent with the formation of these complexes at the cell surface and their internalization into ENs.

In 3T3-L1 adipocytes, insulin induced the association of PI3-kinase mainly with LDM (19) or microsomes (20) whereas PDGF stimulated recruitment and activation of PI 3-kinase exclusively in PM. Correspondingly, only insulin induced GLUT4 translocation from LDM to PM (19, 20). It has recently been shown in 3T3-L1 adipocytes that impaired insulin-stimulated GLUT4 translocation, induced by oxidative stress, is associated with the inhibition of redistribution of both IRS1 and activated PI3-kinase between LDM and cytosol, whereas the activity of this enzyme in total lysates was not affected (36). Taken together, these data support the concept that insulin-induced compartmentalization of key signaling molecules is critical for transducing at least some aspects of the insulin response.

We did not detect insulin-induced changes in the levels of cytosolic p85. This does not exclude translocation, but suggests that it is small in proportion to the size of the cytosolic p85 pool or that there is a balance between recruitment from and to membranes. By 30 sec insulin stimulated the association of p85 with IRS1/2 in cytosol, with approximately 40% and 25% bound to IRS1 and IRS2, respectively. This raises the possibility that insulin-induced phosphorylation of cytosolic IRSs, permitting their recruitment of PI 3-kinase, does not occur only in stable membrane-bound complexes, but may derive from transient associations throughout the exoplasmic space of the cell. It also raises the possibility that as much as 30–40% of p85 binds to other docking proteins or is free.

The model, which explains Akt activation by growth factors, posits Akt translocation to membranes and subsequent activation by phosphorylation. Previous studies have shown that the pleckstrin homology domain of Akt can bind tightly to vesicles containing 3-phosphoinositides (22, 23), and myristylated/palmitylated-Akt, which is targeted to PM, resulted in a constitutively active form of Akt (37). Although this supports the proposed model, several studies failed to show insulin-induced translocation of wild-type Akt to membranes (17, 38); however, a study carried out in primary rat adipocytes demonstrated insulin-induced translocation of Akt to partially purified PM (39). In our study insulin induced a rapid and significant translocation of Akt1 to PM within 30 sec and with a time course similar to that of p85 translocation. The similarity of time course obtained with bpv(phen) as well (data not shown) is consistent with the view that the products of PI 3-kinase activation (3'-phosphoinositides) are involved in Akt translocation. Insulin increased the level of Akt1 in ENs to a lesser extent than in PM. Thus, in rat liver insulin induced the rapid association of Akt1 with both PM and endosomal membranes, consistent with a role for this enzyme in both subcellular compartments.

Full activation of Akt1 requires phosphorylation at Ser473 and Thr308 (24). Our data show that Akt1 is rapidly phosphorylated in PM, ENs, and cytosol, and that the level of phosphorylation of Akt1 in PM was higher than that in ENs or cytosol. Despite this, the time coursed of insulin-stimulated Akt1 activity expressed per molecule of Akt1 were comparable in PM and ENs. Thus, the level of activation per phosphorylated Akt1 was significantly higher in ENs than in the other two compartments examined. We ruled out dephosphorylation of Akt1 during the immunoprecipitation procedure, and confirmed the absence of a correlation between the extent of phosphorylation and the activity of Akt1 in PM and ENs. We thus suggest that another factor(s), in addition to Ser473 and Thr308 phosphorylation, is involved in modulating the extent of Akt1 activation.

In a recent report PKC-{zeta} was shown to associate with Akt and reduce its activity (30), suggesting that PKC-{zeta} could modulate Akt activity. However, we were unable to demonstrate PKC-{zeta} in Akt1 immunoprecipitates (data not shown). It has also been shown that in addition to phosphorylation at Ser473 and Thr308, Akt is phosphorylated at Ser124 and Thr450 (24). However, phosphorylation at these sites was not affected by insulin or insulin-like growth factor, suggesting that they do not contribute to modulation of Akt/PKB after insulin treatment (24).

In summary, our data demonstrated that a physiological dose of insulin in rats induces a rapid trafficking of proteins between subcellular compartments. IRS1/2 are associated with activated PI 3-kinase in PM, ENs, and cytosol. In the membrane fractions the IRS proteins seem to be the principal activators of PI 3-kinase. In parallel with the recruitment of PI 3-kinase, we observed the recruitment and phosphorylation of Akt1 in PM and ENs, in keeping with the view that Akt undergoes translocation to membranes in response to insulin. Our data suggest that another factor(s), in addition to Ser473 and Thr308 phosphorylation, modulates Akt1 activity. Finally, this study demonstrates that ENs constitute a compartment where important downstream insulin signaling molecules concentrate, compatible with the view endocytosis is an important step in insulin signaling.


   Acknowledgments
 
We thank Dr. Catherine Mounier for her critical reading of the manuscript.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council and from the National Cancer Institute of Canada, and by the Cleghorn Fund at McGill University and the M. Pollack Foundation of Montreal. Back

Received June 15, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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