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J. Biol. Chem., Vol. 277, Issue 46, 43565-43571, November 15, 2002

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Regulation of Insulin Receptor Function by a Small Molecule Insulin Receptor Activator^*

Celia Pender, Ira D. Goldfine, Vara Prasad Manchem^§, Joseph L. Evans^§, Wayne R. Spevak^§, Songyuan Shi^§, Sandhya Rao^§, Sonia Bajjalieh^§, Betty A. Maddux, and Jack F. Youngren^¶

From the  Mount Zion Medical Center, University of California, San Francisco, California 94143-1616 and ^§ Telik, Incorporated, South San Francisco, California 94080

Received for publication, March 13, 2002, and in revised form, August 9, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In type 2 diabetes mellitus, impaired insulin signaling leads to hyperglycemia and other metabolic abnormalities. TLK19780, a non-peptide small molecule, is a new member of a novel class of anti-diabetic agents that function as activators of the insulin receptor (IR) -subunit tyrosine kinase. In HTC-IR cells, 20 µM TLK19780 enhanced maximal insulin-stimulated IR autophosphorylation 2-fold and increased insulin sensitivity 2-3-fold. In contrast, TLK19780 did not potentiate the action of insulin-like growth factor-1, indicating the selectivity of TLK19780 toward the IR. The predominant effect of TLK19780 was to increase the number of IR that underwent autophosphorylation. Kinetic studies indicated that TLK19780 acted very rapidly, with a maximal effect observed 2 min after addition to insulin-stimulated cells. In 3T3-L1 adipocytes, 5 µM TLK19780 enhanced insulin-stimulated glucose transport, increasing both the sensitivity and maximal responsiveness to insulin. These studies indicate that at low micromolar levels small IR activator molecules can enhance insulin action in various cultured cells and suggest that this effect is mediated by increasing the number of IR that are tyrosine-phosphorylated in response to insulin. These studies suggest that these types of molecules could be developed to treat type 2 diabetes and other clinical conditions associated with insulin resistance.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cellular response to insulin is mediated through the insulin receptor (IR),¹ a tetrameric protein consisting of two identical extracellular -subunits that bind insulin and two identical transmembrane -subunits that have intracellular tyrosine kinase activity (1, 2). When insulin binds to the -subunit of the receptor, the -subunit tyrosine kinase is activated, resulting in autophosphorylation of -subunit tyrosine residues (3). After autophosphorylation, the activated receptor phosphorylates endogenous substrates, such as the insulin receptor substrates, IRS-1 and IRS-2 (4, 5). Phosphorylated tyrosine residues on these substrates then bind to a variety of other substrates, and insulin action ensues (1-6). It is well established that insulin signaling, including activation of IR tyrosine kinase activity, is impaired in most patients with type 2 diabetes (7, 8).

Pharmacological agents that enhance IR -subunit tyrosine kinase activity could be useful in the treatment of type 2 diabetes (11, 12). Recently, we described a small non-peptide molecule, TLK16998, that interacts with the IR -subunit and enhances insulin action in cultured cells (13). By employing HTC-IR cells (rat hepatoma cells transfected with and overexpressing modest amounts of human IR (14)) under several conditions where IR signaling was impaired, this compound was able to overcome defects in IR signaling (15). Therefore, compounds of this class may be important as anti-diabetic agents.

It would be important to understand the dynamics of how this class of molecules enhances the action of insulin on its receptor. Recently, second generation compounds of this class have been identified that are smaller, more potent, and orally available (16). In the present study, we have employed HTC-IR cells to study how these compounds influence insulin action on the IR. We have determined that they act to increase the number of IR that are tyrosine-phosphorylated in response to insulin.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Compounds and Antibodies-- TLK19780 (Fig. 1) was synthesized by Telik, Inc. (South San Francisco, CA). This compound was screened for IR potency by employing an in vitro IR kinase assay (13). Insulin receptor cytoplasmic tyrosine kinase domain (IR-CKD) was purchased from Stratagene (La Jolla, CA). Monoclonal anti-IR -subunit antibody, MA 20, was produced as described (17). Biotin-conjugated anti-phosphotyrosine antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Site-specific anti-IR antibodies were purchased from BioSource International (Camarillo, CA).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Chemical structure of TLK19780.

Autophosphorylation of IR-CKD-- 200 ng of IR-CKD "-insulin receptor kinase," which is the entire cytoplasmic domain of the IR without any extracellular or transmembrane domain residues, was dissolved in 30 µl of 50 mM Tris (pH 7.4), 2 mM MnCl₂, 10 mM MgCl₂ and combined with increasing concentrations of TLK19780. Reactions were started with the addition of 50 µM ATP (10 µCi of [-³²P]ATP) and incubated for 10 min at 25 °C. For gel analysis, samples were boiled in SDS-PAGE sample buffer for 5 min, electrophoresed on 10% SDS-PAGE gels, and visualized by autoradiography. Radioactivity was quantified by scintillation counting of the labeled bands.

Cell Culture-- HTC-IR (rat hepatoma cells overexpressing human IR, ~2 × 10⁵ receptors/cell) were produced as described previously (18, 19) and cultured in Dulbecco's modified Eagle's medium containing 1.0 g/liter glucose supplemented with 10% fetal calf serum. Penicillin (10 units/ml), fungizone (0.25 µg/ml), and streptomycin (10 µg/ml) were routinely added to cultures, and all the cell lines were cultivated at 37 °C in a 5% CO₂-enriched, humidified atmosphere. 3T3-L1 fibroblasts (ATCC CL-173) were induced to differentiate into adipocytes by incubation in medium containing 1 µM dexamethasone, 0.5 mM isobutylmethylxanthine, and 1.7 µM insulin for 72 h. The cells were switched to medium containing 1.7 µM insulin but without dexamethasone or isobutylmethylxanthine for 48 h. Finally, the cells were returned to normal serum-supplemented medium for 4 days and used to measure glucose transport (see below) 8-13 days post-differentiation. Chinese hamster ovary cells overexpressing human IGF-1 R (CHO-IGF-1 R cells), ~2 × 10⁵ receptors/cell, were a kind gift of Richard Roth (Stanford University) and were cultured in Ham's F-12 medium.

Insulin Stimulation and Treatment of Cells-- Cells were grown in 12-well plates until confluent and then serum-starved for 2 h before preincubation with the compounds. TLK19780 stock solution was prepared by dissolving in Me₂SO. Final concentration of Me₂SO in treated and control cells was 0.1% (v/v). Treatment of control cells in these studies with Me₂SO concentrations up to 0.5% had no effect on IR autophosphorylation (data not shown). In general, cells were preincubated with TLK19780 or Me₂SO (control cells) and stimulated with insulin for 5 min. The reaction was then stopped, and the cells were solubilized. Routinely, preincubation with TLK19780 was for 5 min at 37 °C but was varied for some studies with preincubation up to 60 min with no difference in its effect. Insulin stimulation was performed at 37 °C for 0-60 min with 0-1 µM insulin concentrations with 0.01% (w/v) bovine serum albumin (BSA; Sigma). The reaction was stopped by washing the cells 3 times with ice-cold phosphate-buffered saline (PBS). The cells were solubilized in lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 2 mM sodium orthovanadate) for 60 min at 4 °C. After removal of cellular debris (15,000 × g for 30 min at 4 °C), the protein content in each sample was measured using the Pierce bicinchoninic acid protein assay reagent according to the manufacturer's instructions (Pierce).

Washout Studies-- Studies were conducted to determine the requirement for TLK19780 at the time of insulin incubation and to study the kinetics of IR dephosphorylation following removal of insulin. To examine the requirement for TLK19780 at the time of insulin incubation, 2 sets of cells were preincubated with 20 µM TLK19780 for 10 min at 37 °C. In control cells, 10 nM insulin, 0.01% BSA was added for 5 min at 37 °C. In the other set of cells, TLK19780 was removed by aspirating the media and replacing it with serum-free media. Insulin was then added as for the control cells. Cells were then washed three times with ice-cold phosphate-buffered saline and solubilized in lysis buffer. IR autophosphorylation was determined by ELISA.

The following procedure was used to examine the effects of TLK19780 on the kinetics of IR dephosphorylation. Cells grown in 12-well plates were preincubated with 20 µM TLK19780 for 10 min and stimulated with 10 nM insulin for 11 min. Insulin was removed by aspirating the media and replacing it with serum-free media containing 20 µM TLK19780 or Me₂SO. The decay of IR autophosphorylation was measured by incubating the cells for 0-60 min at 37 °C before stopping the reaction. The reaction was stopped by quickly removing the media and immersing the plate in ice-cold PBS. Cells were then washed 2 more times with ice-cold PBS and solubilized in lysis buffer.

ELISA for Whole-cell IR Autophosphorylation-- The ELISA for tyrosine phosphorylation of the IR was performed as described previously (20). Equal amounts of cell lysates (20 µg of protein) were applied to 96-well ELISA plates coated with monoclonal anti-human IR antibody, MA-20. IR from the samples were allowed to bind during an 18-h incubation at 4 °C. Next, the 96-well ELISA plates were washed with TBST (150 mM NaCl, 0.05% Tween 20, 20 mM Tris (pH 7.4)). Biotin-conjugated anti-phosphotyrosine antibody was added to the wells for 2 h at 22 °C. Wells were again washed and then incubated with streptavidin-horseradish peroxidase. Following the addition of the peroxidase substrate, 3,3',5,5'-tetramethylbenzidine, the degree of tyrosine phosphorylation was quantified by determining the absorbance at 451 nm with a plate reader.

ELISA for Whole-cell IGF-1 R Autophosphorylation-- CHO-IGF-1 R cells were preincubated with TLK19780 or vehicle followed by incubation with IGF-1. The cells were then solubilized as described under "Insulin Stimulation and Treatment of Cells." Equal amounts of cell lysates (20 µg of protein) were applied to 96-well ELISA plates coated with monoclonal anti-human IGF-1 R antibody, IR3. The ELISA then proceeded as described for the IR autophosphorylation ELISA.

Immunoprecipitation-- Cell lysates (500 µg of protein) were diluted to 1-ml final volume with lysis buffer and incubated overnight at 4 °C with 2 µg of phosphotyrosine-specific antibody (anti-pY69, BD Biosciences). The antibody was then precipitated using anti-mouse IgG conjugated to protein A-Sepharose (75 µl of 50% slurry, end over end rotation overnight at 4 °C), pelleted by centrifugation, and washed three times with lysis buffer. The pellet was boiled with 40 µl of 2× Laemmli reducing buffer, and 20 µl was used for Western blotting.

Western Blotting-- To determine the content of phosphorylated IR, anti-phosphotyrosine immunoprecipitates were diluted in 2× Laemmli reducing buffer and were subjected to SDS-PAGE, followed by transfer to nitrocellulose membranes (Amersham Biosciences). The membranes were blocked with 5% nonfat milk and then incubated with antibody specific to the insulin receptor (CT-3, NeoMarkers, Fremont, CA). Immunoreactive proteins were visualized with horseradish peroxidase-coupled anti-mouse IgG and developed with enhanced chemiluminescence reagents as instructed by the manufacturer (PerkinElmer Life Sciences). To determine the content of IR phosphorylated on specific tyrosine residues, equal amounts of lysates from cells treated with insulin in the presence or absence of TLK19780 (30 µg of protein) were diluted in 2× Laemmli reducing buffer and were subjected to SDS-PAGE, followed by transfer to nitrocellulose membranes (Amersham Biosciences). The membranes were blocked with 5% nonfat milk and then incubated with antibodies specific for either phosphorylated tyrosine residues 1158 or 1162-1163 of the IR -subunit (Biosource International). Visualization of immunoreactive proteins was as described above. The signals on the blots were quantified by scanning densitometry.

Glucose Transport Assay-- 3T3-L1 adipocytes were serum-starved in medium containing 0.1% BSA for 16 h prior to use. Adipocytes were stimulated with either increasing concentrations of TLK19780 in the absence of insulin or 5 µM TLK19780 plus increasing concentrations of insulin or IGF-1 (Sigma). Treatments were for 30 min followed by incubation with 0.5 µCi/ml 2-deoxy-D-[¹⁴C]glucose (PerkinElmer Life Sciences) for 30 min at 37 °C. The monolayers were rinsed with cold PBS containing 20 mM glucose and lysed. Radioactivity was quantified by scintillation counting.

Statistics-- The effects of the compounds in combination with insulin or across multiple cell lines were examined by a 2-factor analysis of variance. Comparison of multiple treatment conditions in a single cell line was accomplished by single factor analysis of variance. Post hoc analysis was by paired t test when a significant interaction was obtained. Significance was accepted at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of TLK19780 on the Cytoplasmic Kinase Domain of the Insulin Receptor-- TLK19780 is one member of a unique class of compounds originally identified by their ability to increase autophosphorylation of the isolated, naturally expressed human IR (13). Consistent with that result, we report here that TLK19780 directly activated the cytoplasmic domain of the cloned human IR (Fig. 2). An effect was detectable at 1 nM and increased up to the maximum concentration tested at 10 µM.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effect of TLK19780 on autophosphorylation of the isolated IR cytoplasmic kinase domain. The isolated cytoplasmic tyrosine kinase domain of the cloned human IR (IR-CKD), combined with increasing concentrations of TLK19780, was incubated with 50 µM ATP (10 µCi of [-32P]ATP) as described under "Experimental Procedures." Radioactivity incorporated into the IR-CKD was quantified by SDS-PAGE followed by scintillation counting. Data represent means ± S.E. of three independent experiments and have been expressed as a percent of the buffer control.

Effects of TLK19780 on Insulin Receptor Autophosphorylation-- The direct effects of TLK19780 on IR autophosphorylation in HTC-IR cells were investigated (Fig. 3A). In the absence of insulin, TLK19780 had no effect on IR autophosphorylation at concentrations up to 20 µM. At 30 µM, a 2-3-fold direct effect of the compound on this function was detected (p < 0.05), and maximal effects were seen at 100 µM. In subsequent experiments analyzing the IR-sensitizing effects of the compound, TLK19780 was used at 20 µM.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of TLK19780 on the IR. A, direct effects of TLK19780 on IR -subunit autophosphorylation in HTC-IR cells. HTC-IR cells were incubated in the presence or absence of TLK19780 at the indicated concentrations for 5 min at 37 °C. IR -subunit autophosphorylation was measured by ELISA as described under "Experimental Procedures." Values are mean ± S.E. of the OD (×1000) from four experiments. * indicates values significantly greater than basal (p < 0.05). B, effects of TLK19780 on sensitizing the insulin dose-response of IR -subunit autophosphorylation in HTC-IR cells. HTC-IR cells were stimulated with various concentrations of insulin for 5 min at 37 °C with or without pretreatment for 10 min with TLK19780 (20 µM). Cells were lysed, and IR -subunit autophosphorylation was measured by ELISA. Values are mean ± S.E. of the OD expressed as the percentage of maximal stimulation by insulin alone. * indicates values significantly greater than insulin alone (p < 0.05).

In the absence of TLK19780, a detectable effect of insulin on IR autophosphorylation in these cells was observed at 1 nM, half-maximal effects at ~12 nM, and maximal effects between 100 nM and 1 µM (Fig. 3B).

In the presence of 20 µM TLK19780, IR autophosphorylation, as detected by specific ELISA, was significantly enhanced at every concentration of insulin (p < 0.05). The EC₅₀ value for insulin was reduced from ~12 to ~3 nM with TLK19780. At 10 nM insulin, the maximal responsiveness increased 2-fold with TLK19780.

We next investigated whether the ability of TLK19780 to increase IR autophosphorylation acted through an increase in the number of IR that underwent autophosphorylation or an increase in the number of phosphorylated tyrosine residues per IR. To determine the content of tyrosine-phosphorylated IR, cells were stimulated with insulin plus TLK19780, lysed, immunoprecipitated with an anti-phosphotyrosine antibody, and then Western-blotted with an anti-IR antibody. Fig. 4A shows the results obtained with equal amounts of protein from lysates of cells preincubated with Me₂SO or TLK19780 followed by incubation with insulin. The phosphotyrosine antibody immunoprecipitated a significantly greater number of IR from the cells incubated with insulin plus TLK19780, when compared with cells incubated with insulin alone (p < 0.05). This result indicates that the increased IR phosphotyrosine signal from cells incubated with TLK19780 plus insulin, as detected by ELISA, may be predominantly attributed to an increased number of phosphorylated IR molecules.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   TLK19780 increases the number of IR undergoing -subunit autophosphorylation in HTC-IR cells. A, HTC-IR cells were stimulated with insulin (Ins) for 5 min following pretreatment for 10 min with TLK19780 or vehicle. Cell lysates were immunoprecipitated (IP) using a specific phosphotyrosine antibody (-pY69, Transduction Laboratories) and immunoblotted using -IR (CT3, Neomarkers) as described under "Experimental Procedures." Top, representative Western blot. Bottom, values of phosphorylated IR content are mean ± S.E. of OD determined by scanning densitometry. p values represent significant differences between treatments. B, lysates were prepared from cells incubated with insulin for 5 min following 10 min of pretreatment with either TLK19780 or vehicle. Equal amounts of soluble protein from each preparation were used for Western blot with an antibody specific for phosphorylated tyrosine residues 1162/1163 or an antibody specific for phosphorylated tyrosine residue 1158 of the IR. Blots were stripped and reprobed with an antibody directed against the insulin receptor. Top, representative Western blot. Bottom, values of site-specific IR tyrosine phosphorylation are mean OD determined by scanning densitometry.

In addition, we studied IR autophosphorylation with two antibodies, one specific for phosphorylated tyrosine residues 1162/1163 of the IR and one specific for phosphorylated tyrosine residue 1158 (Fig. 4B). These sites are located in the IR autoregulatory domain that is responsible for IR tyrosine kinase activity (2, 3). Western blots using these site-specific anti-phosphotyrosine antibodies were obtained from cell lysates containing equal amounts of protein from cells stimulated with insulin plus or minus TLK19780. The results obtained with each antibody were similar; a greater number of IR were phosphorylated on sites within this domain following treatment with insulin plus TLK19780 than with insulin alone (Fig. 4B).

Time Course of TLK19780 Action-- Preliminary preincubation studies indicated that the effect of TLK19780 on insulin-stimulated IR autophosphorylation was maximal after a 5-min pretreatment (prior to the addition of insulin) and was stable with preincubations up to 1 h (data not shown). The incubation time course of IR autophosphorylation in response to insulin and insulin plus TLK19780 was then investigated. For each condition, the maximal effect was observed at 2 min (Fig. 5A). With insulin alone, the phosphorylation levels began to decay after 5 min reaching approximately half the maximal value at 60 min. In response to insulin plus TLK19780, IR phosphorylation levels maintained a plateau for 10 min and then decayed at a rate similar to that of insulin alone. However, IR phosphorylation levels remained consistently higher in response to insulin plus TLK19780 over the entire range of incubation, and after 60 min IR phosphorylation levels were still higher than half-maximal value.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Studies of TLK19780 time course. A, IR autophosphorylation time course in HTC-IR cells preincubated for 10 min with TLK19780 or vehicle and then stimulated with 10 nM insulin. After various times cells were solubilized, and the ELISA for IR -subunit autophosphorylation was performed as described under "Experimental Procedures." Values are mean ± S.E. of the OD normalized to 100% of maximal insulin value from triplicate experiments. * indicates values significantly greater than insulin alone (p < 0.05). B, TLK19780 added after insulin stimulation. HTC-IR cells were incubated with 10 nM insulin, and after 10 min 20 µM TLK19780 was added. Cells were lysed after various incubation times, and an ELISA for IR -subunit autophosphorylation was performed. Values are mean ± S.E. of the OD normalized to 100% of maximal insulin value from three experiments per condition.

Studies were next carried out to determine the time course of the TLK19780 effect, and whether this compound would still have a sensitization effect if added after insulin had activated IR autophosphorylation (Fig. 5B). Insulin was added to cells, followed 10 min later by the addition of TLK19780. An effect of the compound was observed within 30 s, and a maximal effect was seen at 2 min.

Studies were next carried out to determine whether the compound had to be present concurrently with insulin in order to produce its effect on IR autophosphorylation (Fig. 6). Cells were preincubated for 10 min with or without TLK19780 and then washed to remove the compound, followed by insulin addition. Compared with unwashed cells, in which the presence of TLK19780 enhanced the effect of insulin, washing out the compound resulted in a complete loss of TLK19780's potentiation of the insulin effect. Studies were then carried out to determine the effect of TLK19780 on the decay of the insulin effect. When insulin and TLK19780 were removed from the media of cells stimulated by both compounds, the decay in IR autophosphorylation was rapid and 90% complete by 10 min (Fig. 7). However, re-addition of TLK19780 alone to the incubation media following this washout markedly slowed this decay rate (Fig. 7).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effect of TLK19780 washout on potentiation of insulin-stimulated IR autophosphorylation in HTC-IR cells. In two parallel experiments cells were pretreated with 20 µM TLK19780 for 10 min. In one experiment media containing TLK19780 were washed out. 10 nM insulin was then added in both experiments, and cells were incubated for 5 min. IR autophosphorylation was quantified by ELISA. Values are mean ± S.E. of the OD for three experiments per condition. * indicates values significantly greater than insulin alone (p < 0.05). § indicates values significantly reduced compared with unwashed cells (p < 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Effect of TLK19780 on IR autophosphorylation after insulin washout. In two parallel experiments HTC-IR cells were pretreated with 20 µM TLK19780 and incubated with 10 nM insulin for 10 min. In one experiment, the media containing insulin and TLK19780 was washed and replaced with serum-free media. In the other experiment, the media was washed and replaced with serum-free media containing 20 µM TLK19780. After various subsequent incubation times, cells were lysed, and IR autophosphorylation was quantified by ELISA. Values are mean ± S.E. of the OD from three experiments per condition. * indicates values significantly greater than cells without TLK19780 replaced (p < 0.05).

TLK19780 Potentiates Insulin-stimulated Glucose Transport in 3T3-L1 Adipocytes-- We next wished to determine whether the effect of TLK19780 on the IR had an effect on a classical insulin biological response, such as glucose transport. Because HTC-IR cells do not have an insulin-sensitive glucose transport system, we studied 3T3-L1 adipocytes that possess this function. In these cells, TLK19780 had no significant effect on glucose transport at concentrations up to 20 µM (Fig. 8A). There was a 2-fold, significant effect of the compound at 30 µM. Preincubation with 5 µM TLK19780 enhanced the stimulatory effect of insulin on glucose transport; the compound both decreased the EC₅₀ of insulin from 33 to 20 nM (p < 0.035) and increased the maximal responsiveness by ~30% (Fig. 8B).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Effects of TLK19780 on insulin-independent and insulin-stimulated glucose transport in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were serum-starved and then treated with increasing concentrations of TLK19780 for 30 min in the absence of insulin. Glucose transport was measured as described under "Experimental Procedures." Data points represent means ± S.E. of four independent experiments using triplicate incubations. Glucose transport in adipocytes treated with vehicle (Me2SO) was set to 100% (basal; 0.002 ± 0.0001 pmol/30 min/well), and all other values are expressed as % basal. *, p < 0.05. B, 3T3-L1 adipocytes were serum-starved and then treated with increasing concentrations of insulin (0.3-300 nM) in the absence or presence TLK19780 (5 µM) for 30 min. Data points represent means ± S.E. of four independent experiments using triplicate incubations. Glucose transport in adipocytes treated with 5 µM TLK19780 and 300 nM insulin was set to 100% (maximum, 0.0092 ± 0.0028 pmol/30 min/well), and all other values expressed as % maximum. The EC50 values for insulin-stimulated transport were 33 nM in adipocytes treated with vehicle and 20 nM in adipocytes treated with TLK19780. * indicates values significantly greater than insulin plus vehicle (p < 0.05).

TLK19780 Does Not Potentiate IGF-1 R Action-- Cells were serum-starved and incubated with 540 µM TLK19780 for 5 min, and IGF-1 R autophosphorylation was measured by ELISA. TLK19780 showed no direct agonist effects over the entire concentration range used (data not shown). CHO-IGF-1 R cells were preincubated for 5 min with 20 µM TLK19780 or vehicle and incubated with IGF-1 for 5 min (Fig. 9A). In contrast to the marked effects of TLK19780 on the IR, the compound had no effect on IGF-1-stimulated IGF-1 R autophosphorylation (Fig. 9A). Similarly TLK19780 had no effects on IGF-1-stimulated glucose transport in 3T3 L1 cells (Fig. 9B).

View larger version (20K): [in this window] [in a new window]   Fig. 9.   Lack of effect of TLK19780 on the IGF-1. A, tyrosine kinase. CHO-IGF-1 R cells were stimulated with various concentrations of IGF-1 for 5 min at 37 °C with or without pretreatment for 10 min with TLK19780 (20 µM). Cells were lysed, and IR -subunit autophosphorylation was measured by ELISA. Values are mean ± S.E. of the OD expressed as the percentage of maximal stimulation by insulin alone. B, glucose transport. 3T3-L1 adipocytes were serum-starved and then treated with increasing concentrations of IGF-1 (0.3-100 nM) in the absence or presence TLK19780 (5 µM) for 30 min. Data points represent means ± S.E. of three independent experiments using triplicate incubations. Glucose transport in adipocytes treated with 5 µM TLK19780 and 100 nM insulin was set to 100%, and all other values are expressed as % maximum.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we have observed that TLK19780, at nanomolar concentrations, acts directly on the cytoplasmic kinase domain of the IR, resulting in increased tyrosine kinase activity. This observation suggests that the IR -subunit is the site of action of TLK19780. Additional studies in intact cells indicate that this compound increases the number of IRs that are activated by insulin.

When employing insulin-sensitive cells, we observed that micromolar concentrations of TLK19780 plus insulin activated the IR to a greater degree compared with insulin alone. The original compound in this series, TLK16998, was a larger, more acidic molecule with a molecular mass of ~1240 Da (13). Accordingly, smaller and more active molecules were synthesized and tested for activity in an in vitro (biochemical) IR tyrosine kinase assay (13, 16). Because TLK19780 was more potent than TLK16998 in vitro (13), it was tested here in cellular models. In the present study with HTC-IR cells, we found that TLK19780, at 20 µM (a concentration that had no IR agonist activity), increased maximal insulin-stimulated IR autophosphorylation by more than 2-fold. In contrast, at this same concentration, TLK16998 only increased insulin-stimulated IR autophosphorylation by ~40% (13). Also, in 3T3-L1 cells, TLK19780 was more effective than TLK16998. As with TLK16998, TLK19780 had little or no effect on the related IGF-1 receptor. This observation demonstrates that reducing the size of the molecule can increase the potency of the molecule without a resulting in a loss of specificity toward the IR.

The present studies employed HTC-IR cells, which are rat hepatoma cells transfected with and overexpressing modest amounts of human IR (14). These cells have been used previously to probe insulin action and insulin signaling, and to study IR activators (14, 15, 21). In the present study, we employed these cells for several reasons. First, IR autophosphorylation can readily be measured in these cells. Second, incubation conditions can be manipulated and kinetic analyses performed. Third, comparisons can be made with prior studies of IR activators.

It is important to note that not only did TLK19780 potentiate IR autophosphorylation in HTC-IR cells but, in addition, similarly enhanced the effect of insulin on glucose transport in a classic insulin-sensitive cell, 3T3-L1 adipocytes. Although these cells are not as sensitive to insulin as isolated fat cells, they are very useful for the types of studies carried out here. The experiments with 3T3 L1 adipocytes indicated that the effect of TLK19780 was not cell line-specific and that the effect of TLK19780 to activate the IR was translated into enhanced insulin-mediated glucose transport. Other studies indicated that the effect of the compound did not potentiate IGF-I R action.

The increased tyrosine phosphorylation of IR observed following treatment of HTC-IR cells with TLK19780 plus insulin could be due to either an increase in the amount of tyrosine phosphorylation per receptor or the number of IR that were tyrosine-phosphorylated. Two types of experiments were carried out to address this important question. First, cells were stimulated with insulin in the presence or absence of TLK19780, immunoabsorbed with anti-phosphotyrosine antibody, and Western-blotted with anti-IR antibody. The results of these studies indicated that more IR contained phosphotyrosine when incubated with TLK19780 plus insulin than with insulin alone. In addition, studies were carried out with site-specific anti-phosphotyrosine antibodies that react only with IR phosphorylated on key tyrosine residues 1162/1163 and 1158. Prior investigations have indicated that phosphorylation of these tyrosine residues is necessary for IR activation (2, 3). Unlike the anti-phosphotyrosine antibody there is only one antigenic site for these site-specific antibodies per -subunit; therefore, the intensity of bands observed in immunoblots is directly related to the number of -subunits phosphorylated at that particular site. The results of the present studies indicated that more IR were phosphorylated on these sites when incubated with TLK19780 plus insulin than with insulin alone. Therefore, these data indicate that a major mechanism by which TLK19780 enhances total IR autophosphorylation is by facilitating the tyrosine phosphorylation of additional IR.

The time course of the effects of TLK19780 on the IR autophosphorylation was also investigated. Preincubation studies indicated that TLK19780 needed to be present for less than 5 min to exert its effect on subsequent stimulation of IR autophosphorylation by insulin. To understand more precisely the kinetics of TLK19780, the compound was added to the cell after insulin-stimulated IR autophosphorylation reached its maximal level. These experiments indicated that the compound could act after the addition of insulin, and reached a maximal effect at 2 min.

The requirement for the continued presence TLK19780 to activate the IR after this compound had been added to cells was then studied. If TLK19780 was added to cells for 10 min, and removed prior to the addition of insulin, no effect was observed. This indicates that TLK19780 was needed during the phosphorylation process. However, when cells were stimulated with TLK19780 plus insulin and only the insulin removed, the presence of TLK19780 maintained a higher level of IR autophosphorylation than when insulin and the compound were both removed. These data indicate that TLK19780 can also modify the ability of the cell to "turn off" the insulin receptor signaling mechanism.

For several reasons, we believe that the effects of TLK19780 are not due to inhibition of cellular tyrosine phosphatase activity. Experiments in a cell-free system using the purified IR cytoplasmic kinase domain (thus in absence of a tyrosine phosphatase) showed that TLK19780 enhanced tyrosine kinase activity. Thus TLK19780 can act directly on the IR. Additionally, for both insulin alone and insulin plus TLK19780, there was a rapid parallel increase in IR autophosphorylation followed by a parallel decrease in this function. If TLK19780 were acting solely by inhibiting tyrosine phosphatase activity, one would not expect the rapid increase in IR autophosphorylation that was observed. Rather, one would expect a time course exhibiting a much slower increase in autophosphorylation, followed by a slower rate of IR dephosphorylation. This latter type of time course was not observed however. Therefore, we believe that these data with TLK19780 are consistent with the notion that the compound acts both to expose the IR to autophosphorylation and to maintain IR phosphorylation.

The effect of TLK19780 was highly specific for the IR, because it did not potentiate the effects of the closely related IGF-I receptor on glucose transport. This finding is in agreement with findings using the first generation compound, TLK16998, which also was highly specific for the IR (13), suggesting that these compounds are not acting as phosphatase inhibitors. Because the -subunits of each receptor are similar, it is likely that the conformational change in the IR -subunit induced by insulin binding, as described by Ottensmeyer and co-workers (22), is sufficiently different from the analogous change in IGF-I receptor to enable the IR to be a specific target of this class of IR activators.

At low concentrations, TLK19780 did not directly activate the IR but rather potentiated the effect of insulin. However, at higher concentrations, there was a direct effect of this compound both on IR autophosphorylation and glucose transport.

In summary, we report that a smaller IR activator molecule can be identified that has potent effects on the IR -subunit at the level of IR activation. Because insulin resistance is a major feature of type 2 diabetes mellitus, it is likely that similar agents may eventually prove useful in the treatment of this disease.

	FOOTNOTES

^* This work was supported by the Dr. Jay Gershow Cancer Research Fund and Harris M. Fishbon Fund of the Mount Zion Health System.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: University of California, San Francisco/Mt. Zion Medical Center, Diabetes and Endocrine Research, Box 1616, San Francisco, CA 94143-1616. Tel.: 415-885-7725; Fax: 415-885-3787; E-mail: drjack@itsa.ucsf.edu.

Published, JBC Papers in Press, September 3, 2002, DOI 10.1074/jbc.M202426200

	ABBREVIATIONS

The abbreviations used are: IR, insulin receptor; IR-CKD, IR-cytoplasmic tyrosine kinase domain; CHO, Chinese hamster ovary; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; IGF, insulin-like growth factor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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