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Persistent Activation of Phosphatidylinositol 3-Kinase Causes Insulin Resistance Due to Accelerated Insulin-Induced Insulin Receptor Substrate-1 Degradation in 3T3-L1 Adipocytes¹

Department of Medicine (K.E., N.N., P.M.S., J.M.O.), Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093; Veterans Administration Research Service (J.M.O.), San Diego, La Jolla, California 92161; The Whittier Diabetes Institute (J.M.O.), University of California, San Diego, La Jolla, California 92093; and the Third Department of Medicine (K.E., H.M., Y.N., A.K., R.K.), Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan

Address all correspondence and requests for reprints to: Jerrold M. Olefsky, Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673.

	Abstract

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Recently, we have reported that the overexpression of a membrane-targeted phosphatidylinositol (PI) 3-kinase (p110CAAX) stimulated p70S6 kinase, Akt, glucose transport, and Ras activation in the absence of insulin but inhibited insulin-stimulated glycogen synthase activation and MAP kinase phosphorylation in 3T3-L1 adipocytes. To investigate the mechanism of p110CAAX-induced cellular insulin resistance, we have now studied the effect of p110CAAX on insulin receptor substrate (IRS)-1 protein. Overexpression of p110CAAX alone decreased IRS-1 protein levels to 63 ± 10% of control values. Insulin treatment led to an IRS-1 gel mobility shift (most likely caused by serine/threonine phosphorylation), with subsequent IRS-1 degradation. Moreover, insulin-induced IRS-1 degradation was enhanced by expression of p110CAAX (61 ± 16% vs. 13 ± 15% at 20 min, and 80 ± 8% vs. 41 ± 12% at 60 min, after insulin stimulation with or without p110CAAX expression, respectively). In accordance with the decreased IRS-1 protein, the insulin-stimulated association between IRS-1 and the p85 subunit of PI 3-kinase was also decreased in the p110CAAX-expressing cells, and IRS-1-associated PI 3-kinase activity was decreased despite the fact that total PI 3-kinase activity was increased. Five hours of wortmannin pretreatment inhibited both serine/threonine phosphorylation and degradation of IRS-1 protein. These results indicate that insulin treatment leads to serine/threonine phosphorylation of IRS-1, with subsequent IRS-1 degradation, through a PI 3-kinase-sensitive mechanism. Consistent with this, activated PI 3-kinase phosphorylates IRS-1 on serine/threonine residues, leading to IRS-1 degradation. The similar finding was observed in IRS-2 as well as IRS-1. These results may also explain the cellular insulin-resistant state induced by chronic p110CAAX expression.

	Introduction

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THE INITIAL step of insulin signaling is binding of insulin to its receptor on the cell surface, resulting in receptor autophosphorylation and activation of the receptor tyrosine kinase. Then, activated tyrosine kinase phosphorylates several intermediate proteins, such as insulin receptor substrates IRS-1, 2, 3, and 4 and the adapter protein, Shc (1, 2, 3). Tyrosine-phosphorylated IRSs, Shc, and the insulin receptor itself bind to Src homology 2 (SH2) domain containing proteins, which further propagates downstream signals.

Several lines of evidence demonstrate that IRS-1 plays an important role in mediating insulin signaling. Tyrosine phosphorylation of IRS-1 has been shown to be necessary for normal insulin-mediated cell cycle progression in a variety of cell systems (4, 5). On the other hand, decreased tyrosine phosphorylation is thought to be responsible for insulin resistance. Reducing IRS-1 expression in rat adipose cells by using an antisense ribosome also leads to modest impairment of insulin-stimulated glucose transporter translocation, and the disruption of the IRS-1 gene in mice causes reduced intrauterine growth, elevated fasting insulin levels, and mild insulin resistance.

Recently, we demonstrated that overexpressed membrane-targeted phosphatidylinositol (PI) 3-kinase (p110CAAX) fully stimulated p70S6 kinase, Akt, 2-deoxyglucose uptake, and Ras without insulin stimulation, whereas, wild-type p110 subunit of PI 3-kinase (p110WT) had little or no effect on these downstream effects (6). Surprisingly, p110CAAX completely inhibited insulin-stimulated glycogen synthase activity in 3T3-L1 adipocytes. Furthermore, p110CAAX did not stimulate MAP kinase phosphorylation, despite the fact that it stimulated Ras activation, and insulin had no further effect, compared with a robust MAP kinase stimulation in control cells. Moreover, p110CAAX stimulated IRS-1 serine/threonine phosphorylation and inhibited IRS-1-associated PI 3-kinase activity, suggesting that PI 3-kinase might play an important role as a negative regulator for insulin signaling.

It has been shown that PI 3-kinase contained both a lipid kinase activity and serine/threonine kinase activity, and that IRS-1 was a potential substrate for PI 3-kinase (7). Moreover, it was reported that serine-phosphorylated IRS-1 inhibited insulin receptor tyrosine kinase activity in TNF--treated 3T3-F442A adipocytes and obese animals (8).

In this paper, we studied the effect of p110CAAX on IRS-1, to investigate the mechanism of p110CAAX-induced cellular insulin resistance. We found that p110CAAX overexpression induced IRS-1 degradation by itself and enhanced insulin-induced IRS-1 degradation. We demonstrated a novel role of PI 3-kinase on cellular insulin resistance caused by accelerated IRS-1 degradation in 3T3-L1 adipocytes.

	Materials and Methods

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Materials
Porcine insulin was kindly provided by Eli Lilly & Co. (Indianapolis, IN). IRS-1 antibody, IRS-2 antibody, and p85 antibody were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Phosphotyrosine antibody (PY20) was from Transduction Laboratories, Inc. (Lexington, KY). Antiinsulin receptor antibody and horseradish peroxidase-linked antirabbit and antimouse antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). DMEM and FCS were obtained from Life Technologies, Inc. (Grand Island, NY). XAR-5 film was obtained from Eastman Kodak Co. (Rochester, NY). [-³²P]ATP and [-³²P]deoxycycidine triphosphate were obtained from NEN Life Science Products (Boston, MA). RO31–8220 was from Calbiochem (San Diego, CA). All other reagents and chemicals were purchased from Sigma (St. Louis, MO).

Cell culture
3T3-L1 cells were grown and maintained in DME high-glucose medium containing 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% FCS in a 10% CO₂ environment. The cells were allowed to grow 2 days post confluency, then differentiated as described earlier (9). Before experimentation, the adipocytes were trypsinized and reseeded in the appropriate culture dishes. The Ad-E1A-transformed human embryonic kidney cell line 293 was cultured in DME high-glucose medium containing 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% FCS in a 5% CO₂ environment.

Preparation of recombinant adenovirus
The recombinant adenovirus containing the complementary DNA (cDNA) encoding the p110CAAX was isolated by homologous recombination with two plasmids, pACCMVpLpA (10) and pJM17 (11), described previously (6). The recombinant plasmid, pAC-p110CAAX, and pJM17 were purified and cotransfected into 293 cells. Because 293 cells were originally derived from adenovirus transformation, the missing E1 gene function of pJM17 was provided in trans. The resulting recombinant virus, containing the p110CAAX, was denoted as Ad5-p110CAAX and was replication defective (at least in cells lacking the E1 region of adenovirus), but fully infectious.

Cell treatment
3T3-L1 adipocytes were infected at a multiplicity of infection (MOI) of 40 plaque formation units/cell for 16 h with stocks of either a control recombinant adenovirus (Ad5-ctrl) containing the cytomegalovirus promoter, pUC 18 polylinker, and a fragment of the SV40 genome, or the recombinant adenovirus Ad5-p110CAAX. Infected cells were incubated for 56 h at 37 C in DME high-glucose medium with 2% heat-inactivated serum, followed by incubation in the starvation medium required for the assay. The efficiency of adenovirus-mediated gene transfer was approximately 90%, as measured by immunocytochemistry. The survival of the differentiated 3T3-L1 adipocytes was unaffected by incubation of cells with the different adenovirus constructs, because the total cell protein remained the same in infected or uninfected cells.

Western blotting
3T3-L1 adipocytes, infected with Ad5-p110CAAX or Ad5-ctrl, were starved for 16 h in DME regular glucose medium with 0.05% FCS. The cells were stimulated with 100 ng/ml insulin for 5–60 min at 37 C and lysed in a solubilizing buffer containing 20 µM Tris, 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40, 50 U/ml of aprotinin, 1 mM Na₃VO₄, 1 mM PMSF, 50 mM NaF (pH 7.5) for 30 min at 4 C. The cell lysates were centrifuged to remove insoluble materials. For Western blot analysis, whole-cell lysates (20 µg protein per lane) were denatured by boiling in Laemmli sample buffer containing 100 mM dithiothreitol and resolved by SDS-PAGE. Gels were transferred to nitrocerurose by electroblotting in Towbin buffer containing 0.02% SDS and 20% methanol. For immunoblotting, membranes were blocked and probed with specified antibodies. Blots were then incubated with horseradish peroxidase-linked second antibody followed by chemiluminescence detection, according to the manufacturer’s instructions (Pierce Chemical Co., Rockford, IL).

RNA extraction and Northern blot analysis
The Ad5-p110CAAX or Ad5-ctrl-infected cells were stimulated with or without 100 ng/ml insulin for 60 min and lysed in TRIZOL reagent (Life Technologies, Inc.). After chloroform extraction, total RNA was isolated by isopropanol precipitation. Total RNA (30 µg) was denatured and electrophoresed on a 1% agarose-2% formaldehyde gel and blotted onto a Nytran N membrane. A full-length rat IRS-1 cDNA and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNA were labeled with [-³²P]deoxycycidine triphosphate by random priming. Then the membrane was incubated with specific probes, washed, and visualized by autoradiography.

PI 3-kinase assay
3T3-L1 adipocytes were infected with Ad5-p110CAAX or Ad5-ctrl at 40 MOI for 16 h at 37 C and grown in medium containing heat-inactivated serum (2%) for 56 h. Serum-starved (16 h) cells were incubated in the absence (basal) or presence of insulin (100 ng/ml) for 10 min, washed once with ice-cold PBS, lysed, and subjected to immunoprecipitation (300–500 mg total protein) with antibody to IRS-1 (4 µg) overnight at 4 C. Immune complexes were precipitated from the supernatant with protein A and washed as described previously (12). The washed immune complexes were incubated with phosphatidylinositol (Sigma, St. Louis, MO) and [-³²P]ATP (3000 Ci/mmol) for 10 min at room temperature. Reactions were stopped with 20 ml of 8 N HCl and 160 ml of CHCl₃:methanol (1:1) and centrifuged; and the lower organic phase was removed and applied to a silica gel TLC plate (EM Science, E. Merck, Darmstad, Germany), which had been coated with 1% potassium oxalate. TLC plates were developed in CHCl₃:CH₃OH:H₂O:NH₄OH (60:47:11.3:2), dried, visualized, and quantitated on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

	Results

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Expression of p110CAAX stimulates IRS-1 degradation
Differentiated 3T3-L1 adipocytes were infected with recombinant adenovirus expressing membrane-localized PI 3-kinase (p110CAAX) and a control recombinant adenovirus (ctrl) at 40 MOI for 16 h (h). After 56 h of incubation, the cells were starved for 16 h and stimulated with 100ng/ml insulin for the indicated times. The cells were lysed and then analyzed by SDS-PAGE, followed by Western blotting with anti-IRS-1 antibody (Fig. 1A), antiphosphotyrosine antibody (Fig. 1B), or antiinsulin receptor antibody (Fig. 1C). p110CAAX expression led to a 37 ± 10% decrease in IRS-1 protein level in the basal state. After insulin stimulation, IRS-1 protein in control cells was reduced by 13 ± 15% at 20 min and 41 ± 12% at 60 min. In contrast, p110CAAX overexpression enhanced insulin-induced IRS-1 degradation up to 61 ± 16% at 20 min and 80 ± 8% at 60 min (Fig. 1, A and D). IRS-1 messenger RNA (mRNA) level was not affected by either insulin or p110CAAX overexpression (Fig. 1E). IRS-1 tyrosine phosphorylation level was decreased by prolonged insulin treatment in control and p110CAAX-expressing cells and correlated with the decreased IRS-1 protein level (Fig. 1B). Neither insulin nor p110CAAX affected insulin receptor protein level or receptor tyrosine phosphorylation (Fig. 1, B and C).

View larger version (36K): [in this window] [in a new window]   Figure 1. Expression of p110CAAX stimulates IRS-1 degradation in 3T3-L1 adipocytes. The differentiated 3T3-L1 adipocytes were infected with Ad5-ctrl (ctrl) or Ad5-p110CAAX (p110CAAX) at 40 MOI for 16 h. After 56 h, the cells were starved for 16 h and stimulated with 100 ng/ml of insulin for the indicated time. Then, the cells were lysed, then analyzed by SDS-PAGE, followed by Western blotting with anti-IRS-1 antibody (A), phosphotyrosine antibody (PY20) (B), and antiinsulin receptor antibody (C). In A, B, and C, each Western blot is representative of five independent experiments. IRS-1 protein levels were analyzed by NIN Image, and results are shown in Fig. 1D. The graph shows means ± SEM of five experiments, and the data are expressed as percentage of the basal protein level in control cells (=100%) ( Ad5-ctrl; , Ad5-p110CAAX). Total RNA (30 µg) was electrophoresed, blotted, and hybridized with full-length rat IRS-1 cDNA (IRS-1) and G3PDH cDNA (Fig. 1E).

View larger version (32K): [in this window] [in a new window]   Figure 2. Expression of p110CAAX decreased insulin-induced IRS-1-p85 of PI 3-kinase association and IRS-1-associated PI 3-kinase activity. Differentiated 3T3-L1 adipocytes were infected with Ad5-ctrl (ctrl) or Ad5-p110CAAX (p110CAAX) at 40 MOI in medium containing heat-inactivated serum (2%) for 16 h. After infection, cells were serum-starved (16 h), incubated in the absence or presence of insulin (100 ng/ml) for the indicated time. Total cell lysates (200–300 µg) were subjected to immunoprecipitation with anti-IRS-1 antibody. The washed immunoprecipitates were subjected to SDS-PAGE and immunoblotted with anti-p85 antibody (A). Each Western blot is representative of three independent experiments. The washed immunoprecipitates were assayed for PI 3-kinase activity, with PI as substrate, and the labeled PI-3 phosphate product (PI-3P) was resolved by TLC and visualized by autoradiography. In B, data from a representative experiment is shown. C shows means ± SEM of three experiments, and the data are expressed as percentage of the maximal activity (=100%) observed in insulin-stimulated, Ad5-ctrl infected cells.

View larger version (51K): [in this window] [in a new window]   Figure 3. Effect of wortmannin on IRS-1 degradation in p110CAAX-expressing 3T3-L1 adipocytes. The differentiated 3T3-L1 adipocytes were infected with Ad5-ctrl (ctrl) or Ad5-P110CAAX (p110CAAX) at 40 MOI for 16 h. After infection, cells were serum starved (16 h), pretreated with 1 µM (A) or 300 nM (B) wortmannin (wort.) for 1 h (lanes 3 and 7) or 5 h (lanes 4 and 8) at 37 C, then stimulated with 100ng/ml insulin for 1 h. The cells were lysed, and subjected to SDS-PAGE, and immunoblotted with anti-IRS-1 antibody. Each Western blot is representative of three independent experiments.

View larger version (45K): [in this window] [in a new window]   Figure 4. Effect of inhibitors on IRS-1 degradation in 3T3-L1 adipocytes. The differentiated 3T3-L1 adipocytes were infected with Ad5-ctrl (ctrl) or Ad5-p110CAAX (p110CAAX) at 40 MOI for 16 h. After 56 h, the cells were starved for 16 h, pretreated with or without 50 µM BAPTA-AM (BAPTA) for 30 min (A, lanes 4, 5, 7, and 8) or 10 µM RO31–8220 (RO) for 15 min (B, lanes 3, 4, 7, and 8), and stimulated with 100 ng/ml insulin for 1 h. Then, the cells were lysed, then analyzed by SDS-PAGE, followed by Western blotting with anti-IRS-1 antibody. Each Western blot is representative of three independent experiments.

View larger version (28K): [in this window] [in a new window]   Figure 5. Expression of p110CAAX stimulates IRS-2 degradation in 3T3-L1 adipocytes. The differentiated 3T3-L1 adipocytes were infected with Ad5-ctrl (ctrl) or Ad5-p110CAAX (p110CAAX) at 40 MOI for 16 h . After 56 h, the cells were starved for 16 h and stimulated with 100ng/ml of insulin for 60 min. Then, the cells were lysed, and analyzed by SDS-PAGE, followed by Western blotting with anti-IRS-2 antibody. Each Western blot is representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It has also been shown that PI 3-kinase contained both a lipid kinase activity and serine/threonine kinase activity, and that IRS-1 was a potential substrate for PI 3-kinase (7). Others have shown that chronic insulin treatment of 3T3-L1 adipocytes led to a decrease in IRS-1 protein concentration by augmenting IRS-1 degradation rate, with little change in the steady-state levels of IRS-1 mRNA (16).

In the current studies, we assessed whether p110CAAX expression compromises IRS-1 function in 3T3-L1 adipocytes. Our studies showed that insulin treatment of cells led to a prominent gel mobility shift of IRS-1, most likely attributable to serine/threonine phosphorylation. This gel shift was followed by subsequent acceleration of IRS-1 degradation, and both effects of insulin were blocked by treatment of the cells with wortmannin. We also found that expression of p110CAAX led to a modest mobility shift of IRS-1 protein, with a more marked effect to down-regulate the cellular content of IRS-1. The effects of p110CAAX expression and insulin were additive, and insulin led to a much more marked decline in IRS-1 protein levels in the p110CAAX-expressing cells. IRS-1 mRNA level was not affected by p110CAAX expression, so decreased IRS-1 protein level was attributable to accelerated IRS-1 degradation. The function of IRS-1 was also compromised in the p110CAAX-expressing cells, because a decrease in IRS-1-associated PI 3-kinase activity was observed. The effects of p110CAAX on IRS-1 were blocked by wortmannin treatment and, interestingly, 5 h of wortmannin treatment led to a substantial recovery of IRS-1 protein content. Because IRS-1 protein content was already down-regulated by the p110CAAX expression before treatment with wortmannin, the data suggest that wortmannin inhibits IRS-1 degradation, and that over a 5-h time period, ongoing biosynthesis of IRS-1 is sufficient to largely replenish cellular IRS-1 content. A rather high concentration (1 µM) of wortmannin was needed to see this inhibition. Because p110CAAX is constitutively active, lower concentrations of wortmannin may be inadequate to block its activity. Indeed, in previous reports, 1 µM wortmannin was needed to inhibit the effects of p110¹ and p110 wild-type plus the internal SH2 domain of p85 (17, 18).

We previously showed that MAP kinase was not activated in the p110CAAX-expressing cells (6). However, it was recently reported that serine/threonine phosphorylation of IRS-1 by PKC involved the MAP kinase and phosphorylation of serine 612 in IRS-1 (14, 15). So, we studied whether the PKC-MAP kinase pathway played a role in p110CAAX-induced IRS-1 degradation. It was reported that 10 µM RO31–8220, PKC inhibitor, suppressed the effects of PKC (19, 20, 21), so it seemed that this concentration of the inhibitor was strong enough to block the kinase activity. However, 10 µM RO31–8220 did not block p110CAAX-induced IRS-1 degradation. Moreover, pretreatment of 20 µM PD98059, MEK inhibitor, completely blocked insulin-induced phosphorylation of MAP kinase but did not block p110CAAX-induced IRS-1 degradation (data not shown). Thus, we conclude that PKC-MAP kinase pathway does not involve p110CAAX-induced IRS-1 degradation.

IRS-2 protein also decreased in the p110CAAX-expressing cells at basal, and insulin accelerated the down-regulation of IRS-2 with its gel mobility shift (Fig. 5). So, it seems that PI 3-kinase also regulates IRS-2, as well as IRS-1, and this effect for IRS proteins may be important in insulin signaling. IRS-3 protein was not detected by Western blotting in 3T3-L1 adipocytes (data not shown).

Several reports suggest that serine/threonine phosphorylation and/or degradation of IRS-1 may play an important role in insulin signaling. For example, serine-phosphorylated IRS-1 inhibited insulin receptor tyrosine kinase activity in TNF--treated 3T3-F442A adipocytes and in obese animals (8). In this study, overexpression of p110CAAX induced IRS-1 serine/threonine phosphorylation but did not inhibit insulin receptor tyrosine phosphorylation (Fig. 1B). So, several pathways may be there for negative regulation of insulin signaling through IRS-1. Okadaic acid induced a similar dose-dependent inhibition of the insulin effect on deoxyglucose uptake, PI 3-kinase activation, and IRS-1 tyrosine phosphorylation; and this was linked to IRS-1 serine/threonine phosphorylation in 3T3-L1 adipocytes (7).

Taken together, these results indicate that insulin leads to serine/threonine phosphorylation of IRSs via a PI 3-kinase-dependent mechanism, which leads to subsequent IRSs degradation. p110CAAX expression mimics this effect of insulin, causing an IRS-1 and -2 gel mobility shift, with subsequent depletion of IRS-1 and -2 proteins. It is possible that the p110CAAX-induced state of cellular insulin resistance (6) is secondary to decreased IRS proteins expression.

	Acknowledgments

 
We thank Dr. Julian Downward for providing p110CAAX cDNA. We also thank Dr. Shu Yamada for technical support.

	Footnotes

 
¹ This work was funded by NIH Research Grant R01-DK-36651 (to J.M.O.) and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to H.M.).

Received October 12, 1999.

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