A Rapamycin-Sensitive Pathway Down-Regulates Insulin Signaling via Phosphorylation and Proteasomal Degradation of Insulin Receptor Substrate-1 (original) (raw)

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1First Department of Medicine (T.H., T.U., J.K., A.T., M.K.) Toyama Medical and Pharmaceutical University Toyama, 930-0194, Japan

*Address requests for reprints to: Tetsuro Haruta, MD, Ph.D., First Department of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama, 930-0194, Japan.

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1First Department of Medicine (T.H., T.U., J.K., A.T., M.K.) Toyama Medical and Pharmaceutical University Toyama, 930-0194, Japan

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1First Department of Medicine (T.H., T.U., J.K., A.T., M.K.) Toyama Medical and Pharmaceutical University Toyama, 930-0194, Japan

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1First Department of Medicine (T.H., T.U., J.K., A.T., M.K.) Toyama Medical and Pharmaceutical University Toyama, 930-0194, Japan

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2Department of Medicine (K.E., P.M.S., J.M.O.) Division of Endocrinology and Metabolism and Whittier Diabetes Institute University of California, San Diego La Jolla, California 92093

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2Department of Medicine (K.E., P.M.S., J.M.O.) Division of Endocrinology and Metabolism and Whittier Diabetes Institute University of California, San Diego La Jolla, California 92093

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2Department of Medicine (K.E., P.M.S., J.M.O.) Division of Endocrinology and Metabolism and Whittier Diabetes Institute University of California, San Diego La Jolla, California 92093

3Veterans Administration Research Service (J.M.O.) San Diego, California 92161

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1First Department of Medicine (T.H., T.U., J.K., A.T., M.K.) Toyama Medical and Pharmaceutical University Toyama, 930-0194, Japan

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12 October 1999

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07 February 2000

Accepted:

10 February 2000

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Tetsuro Haruta, Tatsuhito Uno, Junko Kawahara, Atsuko Takano, Katsuya Egawa, Prem M. Sharma, Jerrold M. Olefsky, Masashi Kobayashi, A Rapamycin-Sensitive Pathway Down-Regulates Insulin Signaling via Phosphorylation and Proteasomal Degradation of Insulin Receptor Substrate-1, Molecular Endocrinology, Volume 14, Issue 6, 1 June 2000, Pages 783–794, https://doi.org/10.1210/mend.14.6.0446
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Abstract

Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor and acts as a docking protein for Src homology 2 domain containing signaling molecules that mediate many of the pleiotropic actions of insulin. Insulin stimulation elicits serine/threonine phosphorylation of IRS-1, which produces a mobility shift on SDS-PAGE, followed by degradation of IRS-1 after prolonged stimulation. We investigated the molecular mechanisms and the functional consequences of these phenomena in 3T3-L1 adipocytes. PI 3-kinase inhibitors or rapamycin, but not the MEK inhibitor, blocked both the insulin-induced electrophoretic mobility shift and degradation of IRS-1. Adenovirus- mediated expression of a membrane-targeted form of the p110 subunit of phosphatidylinositol (PI) 3-kinase (p110CAAX) induced a mobility shift and degradation of IRS-1, both of which were inhibited by rapamycin. Lactacystin, a specific proteasome inhibitor, inhibited insulin-induced degradation of IRS-1 without any effect on its electrophoretic mobility. Inhibition of the mobility shift did not significantly affect tyrosine phosphorylation of IRS-1 or downstream insulin signaling. In contrast, blockade of IRS-1 degradation resulted in sustained activation of Akt, p70 S6 kinase, and mitogen-activated protein (MAP) kinase during prolonged insulin treatment. These results indicate that insulin-induced serine/threonine phosphorylation and degradation of IRS-1 are mediated by a rapamycin-sensitive pathway, which is downstream of PI 3-kinase and independent of ras/MAP kinase. The pathway leads to degradation of IRS-1 by the proteasome, which plays a major role in down-regulation of certain insulin actions during prolonged stimulation.

INTRODUCTION

The insulin receptor mediates insulin action through tyrosine phosphorylation of endogenous substrates, which can recruit Src homology 2 (SH2) domain containing signaling molecules such as the p85-regulatory subunit of phosphatidylinositol (PI) 3-kinase, Grb2, Nck, Crk, Fyn, SHP-2, and others (1, 2). The PI 3- kinase pathway and the ras/mitogen-activated protein (MAP) kinase pathway represent two major elements of insulin receptor signaling, which include multiple protein serine/threonine (Ser/Thr) kinases (1, 2). One of the most important actions of insulin is to enhance incorporation of nutrients into cells. For example, insulin stimulates glucose incorporation into target cells such as skeletal muscle, heart muscle, and adipose cells (1, 2). System A amino acid transport, glycogen synthesis, antilypolysis, and inhibition of gluconeogenesis are also stimulated by insulin (3–8). Insulin also promotes protein synthesis, in which the mammalian target of rapamycin (mTOR), also termed FKBP-rapamycin-associated protein (FRAP) or rapamycin and FKBP12 targets 1 (RAFT1), plays an important role (9). mTOR controls two translational components, eIF-4E binding protein 1 (4E-BP1) and p70 S6 kinase. Phosphorylation of 4E-BP1 upon stimulation releases eukaryotic initiation factor 4E (eIF-4E), allowing it to form a productive initiation complex (9). p70 S6 kinase functions to increase translation of 5′- terminal oligopyrimidine tract mRNAs, which largely code for ribosomal proteins and other elements of the translational machinery (9).

Insulin receptor substrate-1 (IRS-1) is an endogenous substrate of the insulin receptor and contains at least 20 potential tyrosine phosphorylation sites and more than 30 potential Ser/Thr phosphorylation sites (10). Even in the basal state, IRS-1 is highly phosphorylated on Ser and Thr residues, which explains the migration of IRS-1 on SDS-PAGE at an electrophoretic mobility corresponding to approximately 185 kDa, compared with its predicted molecular mass of 131 kDa (10). Insulin stimulation immediately elicits IRS-1 phosphorylation on both tyrosine and Ser/Thr residues (11) and subsequently produces a shift in mobility on SDS-PAGE due to further Ser/Thr phosphorylation (11).

Recent studies in cultured cells, animal models, and human disease states have suggested that Ser/Thr phosphorylation of IRS-1 may be involved in the development of insulin resistance (12–25). Several studies have attempted to identify a Ser/Thr kinase responsible for phosphorylation of IRS-1 that leads to attenuation of insulin signaling. For example, glycogen synthase kinase-3 was reported to phosphorylate IRS-1 and inhibit insulin receptor tyrosine kinase activity (20). Activation of protein kinase C by phorbol 12-myristate-13-acetate elicits Ser phosphorylation of IRS-1, which is mediated by MAP kinase and leads to a decreased ability of the insulin receptor to tyrosine-phosphorylate IRS-1 with decreased p85 association (19). Platelet-derived growth factor (PDGF) stimulation causes an IRS-1 mobility shift due to Ser/Thr phosphorylation, and this effect is mediated through PI 3-kinase (23, 24). More recently, Akt (also called protein kinase B) was found to mediate Ser/Thr phosphorylation of IRS-1, and this was inhibited by rapamycin (25). However, the molecular mechanisms and the functional consequences of insulin-induced Ser/Thr phosphorylation of IRS-1 are largely unknown.

Furthermore, earlier studies indicated that chronic exposure of cells to insulin resulted in degradation of IRS-1 protein (26–30). The rate of IRS-1 degradation was about 10 times faster in insulin-treated cells than in basal cells (26). Although a role for PI 3-kinase in insulin-induced degradation of IRS-1 was suggested (28), little is known about the mechanism and the role of insulin-induced degradation of IRS-1, and the relationship between phosphorylation and degradation of IRS-1 has not been investigated.

In the present study, we have explored the molecular mechanisms of insulin-stimulated Ser/Thr phosphorylation and degradation of IRS-1, as well as the functional consequences. We show here that a rapamycin-sensitive pathway is involved in both of these phenomena, which eventually mediates degradation of IRS-1 by the proteasome. Our data suggest a role for this pathway in down-regulation of insulin action during prolonged stimulation.

RESULTS

Insulin Induces an Electrophoretic Mobility Shift and Degradation of IRS-1

To investigate the molecular mechanism of the insulin-induced mobility shift and degradation of IRS-1, we first determined the time course of these effects, as well as tyrosine phosphorylation of IRS-1 and the phosphorylation status of molecules that mediate insulin action. Fully differentiated 3T3-L1 adipocytes were stimulated with 20 nm insulin for various periods, and cell lysates were subjected to SDS-PAGE followed by immunoblotting with anti-IRS-1, phosphotyrosine, phospho-specific Akt, phospho-specific p70 S6 kinase, or phospho-specific MAP kinase antibodies (Fig. 1). As reported previously (11), insulin stimulation increased the apparent molecular mass of IRS-1 in a time-dependent manner with the maximal gel mobility shift observed at 1 h after stimulation (Fig. 1). When the cells were stimulated for longer time periods, the mobility of IRS-1 gradually returned to the basal state, and the intensity of the IRS-1 band decreased in a time-dependent manner, indicating that IRS-1 protein was gradually degraded, as previously shown (26–30). IRS-1 protein levels decreased to 31% and 12% of the original level at 4 and 8 h after insulin stimulation, respectively. Even at 1 h after insulin stimulation, IRS-1 protein level was decreased to 75% (Fig. 1). At 24 h after stimulation with insulin, the IRS-1 protein level was somewhat recovered compared with that at 8 h after insulin stimulation (Fig. 1). Tyrosine phosphorylation of pp185, most of which is IRS-1 in 3T3-L1 adipocytes (31), was induced rapidly in response to insulin, with a maximum at 1–5 min after insulin stimulation, a gradual decrease thereafter, and a slight recovery at 24 h (Fig. 1). Thus, the time course of pp185 tyrosine phosphorylation apparently correlated with IRS-1 protein levels. However, densitometric analysis showed that pp185 levels decreased only to 57% and 37% of the original level at 4 and 8 h after insulin stimulation, respectively (Fig. 1), indicating that the decrease of pp185 levels is less than that of IRS-1 protein levels. The difference may be due to a continuous increase in tyrosine phosphorylation of IRS-1 and/or preferential degradation of IRS-1 molecules that are not phosphorylated on tyrosine residues. Phosphorylation of Akt on Ser473 was rapidly induced at 1 min after stimulation with insulin, remained stable for 20–60 min, and then gradually decreased (Fig. 1). A similar time course was observed for phosphorylation of Thr308 of Akt (Fig. 1). Phosphorylation of p70 S6 kinase on Ser411 was induced more slowly, reaching a maximum at 20–60 min after insulin stimulation, decreasing slowly thereafter (Fig. 1). Phosphorylation of Thr389 of p70 S6 kinase showed a similar time course (Fig. 1). Phosphorylation of MAP kinase was rapidly stimulated and was maximal at 5 min after insulin stimulation and gradually decreased thereafter (Fig. 1).

Fig. 1.

Time Course of Electrophoretic Mobility Shift and Degradation of IRS-1 Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h and stimulated with 20 nm insulin for indicated periods of time. Cell lysates were then analyzed by electrophoresis and immunoblotting with anti-IRS-1, antiphosphotyrosine, antiphospho-Ser473 Akt, antiphospho-Thr308 Akt, antiphospho-Ser411 p70 S6 kinase, antiphospho-Thr389 p70 S6 kinase, or antiphospho-Thr202/Tyr204 MAP kinase antibody. Protein amounts of Akt, p70 S6 kinase, and MAP kinase as assessed by immunoblotting with non-phospho-specific antibodies were not affected during the period examined (data not shown). Data were analyzed by densitometry and means ± se of three independent experiments are shown (lower panels).

PI 3-Kinase Inhibitors and Rapamycin, but Not the MEK Inhibitor Block the Insulin-Induced Mobility Shift of IRS-1

To determine which insulin signaling pathway mediates the insulin-induced mobility shift of IRS-1, we examined the effects of PI 3-kinase inhibitors, rapamycin (which inhibits mTOR signaling pathway), or the MEK inhibitor. Since maximal mobility shift was observed at 60 min after insulin stimulation (Fig. 1), cells were stimulated with insulin for 60 min after pretreatment with various concentrations of each inhibitor. The mobility of IRS-1 and the phosphorylation status of downstream kinases were monitored by immunoblot analysis of cell lysates. Pretreatment of the cells with a PI 3-kinase inhibitor, wortmannin, inhibited the insulin-induced mobility shift of IRS-1 in a dose- dependent manner, along with a dose-dependent inhibition of phosphorylation of both Akt and p70 S6 kinase (Fig. 2A). A structurally different PI 3-kinase inhibitor, LY294002, had similar effects (Fig. 2B). Pretreatment of the cells with rapamycin also resulted in a dose-dependent inhibition of the insulin-induced mobility shift of IRS-1 (Fig. 2C), along with a dose-dependent inhibition of phosphorylation of p70 S6 kinase, but not phosphorylation of Akt (Fig. 2C). The MEK inhibitor, PD98059, had no significant effect on the IRS-1 mobility or phosphorylation of Akt, and only a slight effect on phosphorylation of p70 S6 kinase, while it inhibited insulin-induced MAP kinase phosphorylation in a dose-dependent manner (Fig. 2D).

Fig. 2.

Effects of PI 3-Kinase Inhibitors, Rapamycin or PD98059, on Insulin-Induced Electrophoretic Mobility Shift of IRS-1 Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h and pretreated with indicated concentrations of wortmannin (panel A), LY294002 (panel B), rapamycin (panel C), PD98059 (panel D) or vehicle[ dimethylsulfoxide (DMSO), final 0.1%] for 30 min before stimulation without or with 20 nm insulin for 60 min. Cell lysates were then analyzed by electrophoresis and immunoblotting with anti-IRS-1, antiphospho-Ser473 Akt, antiphospho-Ser411 p70 S6 kinase, or antiphospho-Thr202/Tyr204 MAP kinase antibody. None of the treatments affected protein amounts of Akt, p70 S6 kinase, or MAP kinase as assessed by immunoblotting with non-phospho-specific antibodies (data not shown). A representative of three independent experiments is shown.

PI 3-Kinase Inhibitors and Rapamycin, but Not the MEK Inhibitor Prevent Insulin-Induced Degradation of IRS-1

It has been previously reported that insulin-induced IRS-1 degradation is mediated by a PI 3-kinase- dependent pathway (28). Therefore, to evaluate this, we examined the effects of the inhibitors on insulin- induced degradation of IRS-1 and tyrosine phosphorylation of pp185. Wortmannin almost completely inhibited insulin-induced degradation of IRS-1 at 4 h after insulin stimulation at the same concentration that inhibited phosphorylation of Akt and p70 S6 kinase (Fig. 3A). The insulin-induced mobility shift (at 1 h) was also inhibited (Fig. 3A). The decrease in tyrosine phosphorylation of pp185 was also inhibited in parallel (Fig. 3A). Another PI 3-kinase inhibitor, LY294002, had similar effects (Fig. 3B). Pretreatment of the cells with rapamycin, at a concentration that inhibited the insulin-induced mobility shift, and phosphorylation of p70 S6 kinase also clearly inhibited IRS-1 degradation at 4 h after insulin stimulation (Fig. 3C). Tyrosine phosphorylation of IRS-1 again appeared to correlate with IRS-1 protein levels (Fig. 3C). PD98059 had no effect on degradation or tyrosine phosphorylation of IRS-1 (Fig. 3D). Taken together, the above results indicate that a rapamycin-sensitive pathway, which is downstream of PI 3-kinase, but not the ras/MAP kinase pathway, is required for both insulin-induced Ser/Thr phosphorylation and degradation of IRS-1.

Fig. 3.

Effects of PI 3-Kinase Inhibitors, Rapamycin or PD98059, on Insulin-Induced Degradation and Tyrosine Phosphorylation of pp185 Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h and pretreated with indicated concentrations of wortmannin (panel A), LY294002 (panel B), rapamycin (panel C), PD98059 (panel D), or vehicle (DMSO, final 0.1%) for 30 min before stimulation without or with 20 nm insulin for 1 or 4 h. Cell lysates were then analyzed by electrophoresis and immunoblotting with anti-IRS-1, antiphosphotyrosine, antiphospho-Ser473 Akt, or antiphospho-Ser411 p70 S6 kinase antibody. None of the treatments affected protein amounts of Akt, p70 S6 kinase, or MAP kinase as assessed by immunoblotting with non-phospho-specific antibodies (data not shown). Data were analyzed by densitometry and means ± se of three independent experiments are shown (right panels).

Overexpression of p110CAAX Induces a Mobility Shift and Degradation of IRS-1 via a Rapamycin-Sensitive Pathway

Fully differentiated 3T3-L1 adipocytes were infected with the adenovirus vector, Ad5-p110CAAX, which expresses a membrane-targeted form of the catalytic subunit of PI 3-kinase. At 48 h after infection, cell lysates were subjected to SDS-PAGE and immunoblot analysis. Ad5-p110CAAX caused phosphorylation of both Akt and p70 S6 kinase (Fig. 4A), indicating that membrane targeting of the p110 subunit led to activation of these downstream effector molecules of PI 3-kinase, as shown previously (32). Ad5-p110CAAX induced the mobility shift of IRS-1 (Fig. 4A), although the extent of this was less than that induced by insulin (data not shown). Since the insulin-induced mobility shift declines after 1 h (Fig. 1), the relatively small mobility shift induced by p110CAAX may be due to continuous activation of PI 3-kinase. The results also show that p110CAAX caused a greater loss of IRS-1 protein (Fig. 4A), indicating that IRS-1 was degraded as a result of overexpression of p110CAAX. We examined the effect of rapamycin on the p110CAAX-induced IRS-1 mobility shift and degradation. As can be seen in Fig. 4B, the mobility shift of IRS-1 induced by p110CAAX was inhibited by rapamycin in a dose- dependent manner. The loss of IRS-1 protein was also partially restored by rapamycin (Fig. 4B). Rapamycin also inhibited the p110CAAX-induced phosphorylation of p70 S6 kinase in a dose-dependent manner without any effect on Akt phosphorylation (Fig. 4B). MAP kinase phosphorylation was not stimulated by p110CAAX overexpression and was not affected by rapamycin (Fig. 4B).

Fig. 4.

Effects of p110CAAX and Rapamycin on Electrophoretic Mobility and Protein Amounts of IRS-1 Fully differentiated 3T3-L1 adipocytes were infected with either Ad5-CT or Ad5-p110CAAX at the indicated multiplicity of infection (m.o.i.). At 48 h after infection, cells were serum starved for 16 h (panel A) or cells were treated with indicated concentrations of rapamycin or vehicle (panel B, DMSO, final 0.1%) for 4 h after serum starvation for 16 h. Cell lysates were analyzed by electrophoresis and immunoblotting with anti-IRS-1, antiphospho-Ser473 Akt, antiphospho-Ser411 p70 S6 kinase or antiphospho-Thr202/Tyr204 MAP kinase antibody. Neither infection of each adenovirus vector nor treatment with rapamycin had any effect on protein amounts of Akt or p70 S6 kinase as assessed by non-phospho-specific antibodies (data not shown). IRS-1 protein levels were analyzed by densitometry and means ± se of three independent experiments are shown (B, lower panel).

Proteasome Inhibitors Block Insulin-Induced Degradation but Not the Mobility Shift of IRS-1

To explore the mechanism of insulin-induced degradation of IRS-1, we next examined the effects of proteasome inhibitors on insulin-induced mobility shift and degradation of IRS-1 (Fig. 5). Cells were pretreated with lactacystin (33) for 60 min, followed by stimulation with insulin for either 1 or 4 h. Lactacystin almost completely inhibited insulin-induced degradation of IRS-1 at 4 h (Fig. 5). At 1 h after insulin stimulation, the inhibitor did not affect the insulin-induced mobility shift of IRS-1 (Fig. 5). Moreover, the mobility shift did not decrease even at 4 h after insulin stimulation (Fig. 5). Less specific inhibitors for the proteasome, MG132 and proteasome inhibitor I (PSI), had similar effects on IRS-1 degradation and mobility shift (data not shown). Insulin-stimulated p70 S6 kinase phosphorylation was not affected by lactacystin at 1 h and was enhanced at 4 h after stimulation (Fig. 5), indicating that the inhibitory effect of lactacystin on insulin-induced degradation of IRS-1 was not mediated through inhibition of the rapamycin- sensitive pathway, and that inhibition of IRS-1 degradation may augment insulin signaling.

Fig. 5.

Effects of Lactacystin on Electrophoreitc Mobility Shift and Degradation of IRS-1 Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h and pretreated with 10 μm lactacystin or vehicle (DMSO, final 0.1%) for 60 min before stimulation without or with 20 nm insulin for 1 or 4 h. Cell lysates were analyzed by electrophoresis and immunoblotting with anti-IRS-1 or antiphospho-Ser411 p70 S6 kinase antibody. Protein amounts of p70 S6 kinase, as assessed by immunoblotting with non-phospho-specific antibody were not affected by the treatment (data not shown). A representative of three independent experiments is shown.

Rapamycin and Lactacystin Enhance Insulin-Induced Phosphorylation of Akt

To evaluate the functional consequences of the IRS-1 mobility shift and IRS-1 degradation, we determined the effects of rapamycin and lactacystin on downstream events of insulin signaling. First, we tested the effects of these inhibitors on phosphorylation of Akt at either 1 or 4 h after insulin stimulation. At 1 h, both rapamycin and lactacystin increased Akt phosphorylation to a small extent (∼25%) but had larger effects at 4 h (Fig. 6). Since rapamycin inhibited both the electrophoretic mobility shift and degradation of IRS-1, whereas lactacystin inhibited degradation alone, these observations suggest that the effect of rapamycin on Akt phosphorylation may be through inhibition of IRS-1 degradation, rather than inhibition of IRS-1 Ser/Thr phosphorylation.

Fig. 6.

Effects of Rapamycin or Lactacystin on Insulin-Stimulated Phosphorylation of Akt Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h and pretreated with 4 nm rapamycin for 30 min (panel A), 10μ m lactacystin for 60 min (panel B) or vehicle (DMSO, final 0.1%) before stimulation without or with 20 nm insulin for indicated time periods. Cell lysates were analyzed by electrophoresis and immunoblotting with anti-phospho-Ser473 Akt antibody. Protein amounts of Akt as assessed by immunoblotting with non-phospho-specific Akt antibody were not affected by the treatment (data not shown). Immunoblots of three independent experiments were quantitated by a densitometer. The values were corrected for those of control cells stimulated with insulin for 1 h. Means ± se are shown as bar graphs.

Lactacystin Causes Sustained Activation of Insulin Signaling

To assess the role of IRS-1 degradation in insulin action, we examined the effect of lactacystin on insulin signaling during prolonged stimulation. Cells were pretreated with lactacystin and stimulated with insulin for various time periods up to 12 h. IRS-1 protein levels, tyrosine phosphorylation of IRS-1, association of p85 with IRS-1, phosphorylation status and protein levels of Akt, p70 S6 kinase and MAP kinase and protein levels of p85, insulin receptor, and GLUT4 were determined by immunoblot analysis of the cell lysates or anti-IRS-1 antibody immunoprecipitates (Fig. 7). The results show that progressive loss of IRS-1 protein during prolonged insulin stimulation was almost completely inhibited by lactacystin (Fig. 7A). In lactacystin-treated cells, the mobility shift of IRS-1 did not decrease during the stimulation period (Fig. 7A). Lactacystin significantly inhibited the progressive decrease of pp185 tyrosine phosphorylation (Fig. 7A). Similar results were obtained for protein levels and tyrosine phosphorylation of IRS-1 (Fig. 7B). The decrease in the amount of IRS-1-associated p85 was also significantly inhibited by lactacystin (Fig. 7B), while total cellular p85 was not affected by prolonged insulin treatment or by lactacystin (Fig. 7C). The decrease in phosphorylation of Akt, p70 S6 kinase, and MAP kinase during prolonged insulin stimulation, as assessed by immunoblotting with phosphospecific antibodies, was also significantly inhibited by lactacystin (Fig. 7A). The protein amounts of Akt, p70 S6 kinase and MAP kinase, insulin receptor, and GLUT4 did not change during prolonged insulin treatment and were not affected by lactacystin (Fig. 7C). Therefore, the effect of lactacystin on down-regulation of Akt, p70 S6 kinase, and MAP kinase activation during chronic insulin stimulation correlated with the effect on IRS-1 degradation.

Fig. 7.

Effects of Lactacystin on Insulin Signaling during Prolonged Stimulation Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h and pretreated with 10 μm lactacystin or vehicle (DMSO, final 0.1%) for 60 min before stimulation without or with 20 nm insulin for the indicated time periods. A, Cell lysates were analyzed by electrophoresis and immunoblotting with anti-IRS-1, antiphosphotyrosine, antiphospho-Ser473 Akt, antiphospho-Thr308 Akt, antiphospho-Ser411 p70 S6 kinase, antiphospho-Thr389 p70 S6 kinase, or antiphospho-Thr202/Tyr204 MAP kinase antibody. Data were analyzed by denstitometry and means ± se of three independent experiments are shown (lower panels: open symbols, without lactacystin; closed symbols, with lactacystin). B, Cell lysates were immunoprecipitated with anti-IRS-1 antibody, and the immune complexes were then analyzed by electrophoresis and immunoblotting with anti-IRS-1, antiphosphotyrosine, or anti-p85 antibody. Data were analyzed by denstitometry, and means ± se of three independent experiments are shown (lower panels: open symbols, without lactacystin; closed symbols, with lactacystin). C, Protein amounts of Akt, p70 S6 kinase, MAP kinase, p85, insulin receptor (IR), and GLUT4 were assessed by immunoblotting with the indicated non-phospho-specific antibodies. A representative of three independent experiments is shown.

Rapamycin but Not Lactacystin Enhances Insulin-Stimulated 2-Deoxyglucose (2-DOG) Uptake

Finally, we examined the effect of rapamycin or lactacystin on insulin-stimulated 2- DOG uptake. Cells were pretreated with rapamycin or lactacystin and stimulated with insulin for various time periods, followed by measurement of 2-DOG uptake. Rapamycin treatment enhanced 2-DOG uptake by 34% and 52% at 1 and 4 h after insulin stimulation, respectively (Fig. 8). Thus, the effect of rapamycin on 2-DOG uptake was similar to the effects of rapamycin or lactacystin on Akt phosphorylation (Fig. 6). In contrast, lactacystin had no significant effect on insulin-stimulated 2-DOG uptake at any time point examined (Fig. 8).

Fig. 8.

Effects of Rapamycin or Lactacystin on Insulin-Stimulated 2-DOG Uptake Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h and pretreated with vehicle (DMSO, final 0.1%) (circle), 4 nm rapamycin for 30 min (triangle) or 10 μm lactacystin for 60 min (square) before stimulation without or with 20 nm insulin for the indicated time periods.[ 3H]2-DOG uptake was then measured as described in Materials and Methods. Means ± se of six independent experiments are shown (*, P > 0.05; **, P > 0.01).

DISCUSSION

The current results show that a rapamycin-sensitive pathway, downstream of PI 3-kinase, mediates the insulin-induced electrophoretic mobility shift and degradation of IRS-1. This is based on the observation that two structurally unrelated PI 3-kinase inhibitors, as well as rapamycin, inhibited these effects of insulin. Moreover, the finding that activation of the PI 3-kinase pathway by expression of p110CAAX was sufficient to induce both of these phenomena in a rapamycin-sensitive manner further supported the role of PI 3-kinase and places the rapamycin-sensitive pathway downstream of PI 3- kinase. The dependence of the insulin-induced IRS-1 mobility shift on PI 3-kinase and a rapamycin-sensitive pathway agrees with the previous finding that PDGF-induced Ser/Thr phosphorylation of IRS-1 was inhibited by PI 3-kinase inhibitors (24). It is also consistent with the recent observation that attenuation of insulin-stimulated tyrosine phosphorylation of IRS-1 by pretreatment with PDGF or activation of Akt was prevented by rapamycin (25). Therefore, it seems likely that activation of PI 3- kinase either by insulin or PDGF induces Ser/Thr phosphorylation of IRS-1 through a common rapamycin- sensitive pathway, which is possibly mediated by activation of Akt. Rapamycin acts by binding to mTOR as a gain-of-function complex with FKBP12, thus compromising the catalytic activity of mTOR (34, 35). Therefore, mTOR itself, or a Ser/Thr kinase regulated by mTOR, may be responsible for the insulin-induced Ser/Thr phosphorylation of IRS-1. Alternatively, it is possible that a Ser/Thr protein phosphatase that is negatively regulated by mTOR (36) may play a role in dephosphorylation of IRS-1.

Evidence has indicated that Ser/Thr phosphorylation of IRS-1 induced by various agents or mediators inhibits insulin-stimulated tyrosine phosphorylation of IRS-1, leading to attenuation of insulin signaling (12–25). Our data, however, suggest that Ser/Thr phosphorylation of IRS-1 caused by insulin, which is responsible for the insulin-mediated mobility shift, may not significantly affect IRS-1 tyrosine phosphorylation or downstream insulin signaling, since tyrosine phosphorylation of IRS-1 did not seem to be significantly affected by inhibition with PI 3-kinase inhibitors or rapamycin, and the effects of rapamycin and lactacystin on Akt phosphorylation were not significantly different. On the other hand, the insulin-induced mobility shift occurs slowly as compared with tyrosine phosphorylation of IRS-1 (Fig. 1). Therefore, when IRS-1 is already phosphorylated on tyrosine residues, Ser/Thr phosphorylation induced by insulin may not significantly affect tyrosine phosphorylation of IRS-1 or downstream insulin signaling. Thus, the current data are still consistent with the idea that Ser/Thr phosphorylation of IRS-1 induced through a rapamycin-sensitive pathway by other agents or mediators, such as PDGF, Akt, or PI 3-kinase (23–25, 32), may impair insulin-stimulated tyrosine phosphorylation of IRS-1. It is also possible that increases in tyrosine phosphorylation of IRS-1 and downstream insulin effects in response to restimulation with insulin may be attenuated when Ser/Thr phosphorylation of IRS-1 is already induced by insulin.

We have shown that a highly specific inhibitor for the proteasome, lactacystin, as well as less specific inhibitors, almost completely prevented insulin-induced degradation of IRS-1. This result is consistent with the recent report by Sun et al. (37). Furthermore, our finding that insulin-induced degradation of IRS-1 was also dependent on PI 3-kinase and the rapamycin-sensitive pathway strongly suggests that insulin-stimulated Ser/Thr phosphorylation of IRS-1, which seems to be mediated by mTOR, may be required for its subsequent degradation by the proteasome. Consistent with this notion, the data showed that the mobility shift of IRS-1 did not diminish during prolonged insulin treatment in the presence of proteasome inhibitors, suggesting that the proteasome may specifically degrade Ser/Thr-phosphorylated IRS-1. The vast majority of known protein substrates of the proteasome must be modified by the covalent attachment of a polyubiquitin chain, which serves as a substrate-targeting and recognition signal for the proteasome, and, in many instances, signal-induced phosphorylation is a prerequisite for subsequent ubiquitination of the substrate protein (38–41). Therefore, phosphorylation of IRS-1 mediated by the rapamycin-sensitive pathway may lead to ubiquitination of IRS-1, which is required for degradation by the proteasome. Alternatively, Ser/Thr phosphorylation of IRS-1 may lead to modification of IRS-1 other than ubiquitination or promote interaction of the already ubiquitinated or modified IRS-1 with a component of the proteasomal degradation machinery. The recent report that IRS-1 was polyubiquitinated, even in the basal state, and that insulin stimulation was without effect on the extent of the ubiquitination (37) agrees with the latter possibilities.

Our data demonstrate that degradation of IRS-1 by the proteasome plays a major role in down-regulation of insulin signaling during prolonged stimulation. Thus, blockade of IRS-1 degradation by either rapamycin or lactacystin resulted in increases in tyrosine phosphorylation of IRS-1 and significant enhancement of Akt phosphorylation at 4 h after insulin stimulation. Furthermore, blockade of IRS-1 degradation by lactacystin significantly inhibited the progressive decrease of IRS-1-associated p85 and progressive inactivation of Akt, p70 S6 kinase, and MAP kinase until 12 h after insulin stimulation. The finding that chronic insulin treatment or lactacystin were without effect on protein levels of Akt, p70 S6 kinase, and MAP kinase and other components of insulin signaling cascade, including insulin receptor, p85, and GLUT4, further supports the contribution of IRS-1 degradation to down-regulation of insulin action during prolonged stimulation.

Although blockade of IRS-1 degradation by lactacystin enhanced insulin effects such as activation of Akt, p70 S6 kinase, and MAP kinase, lactacystin was without effect on 2-DOG uptake, whereas this was significantly enhanced by rapamycin. The reason for the differential effects of lactacystin and rapamycin on 2-DOG uptake is not clear. One explanation is that IRS-1 may not play a key role in insulin-stimulated glucose transport, consistent with several previous reports (42–45). In this scenario, rapamycin may enhance insulin-stimulated 2-DOG uptake by a mechanism independent of IRS-1. Alternatively, the observation that the mobility shift of IRS-1 did not decline during prolonged insulin stimulation in the presence of lactacystin raises the possibility that inhibition of proteasomal degradation results in the accumulation of modified forms of IRS-1. These forms of IRS-1 may not target to the intracellular location appropriate for eliciting GLUT4 translocation, but still can couple to other effects of insulin including activation of Akt, p70 S6 kinase, and MAP kinase.

mTOR controls protein synthesis by regulating the translational components, 4E-BP1 and p70 S6 kinase (9). Recent evidence suggests that mTOR functions as a sensor of amino acids and balances nutrient supply and protein synthesis (35, 46, 47). Since one of the major metabolic actions of insulin is to stimulate uptake of amino acids, it is tempting to speculate that modulation of insulin action by the rapamycin-sensitive pathway may be a part of an adaptational response to nutrient availability.

In summary, these results indicate that both insulin-stimulated Ser/Thr phosphorylation of IRS-1, which is seen as an electrophoretic mobility shift, and insulin-induced degradation of IRS-1 are mediated by a rapamycin-sensitive pathway that is downstream of PI 3-kinase and independent of the ras/MAP kinase pathway. The results further show that the pathway leads to degradation of IRS-1 by the proteasome, which plays a major role in down-regulation of insulin action during prolonged insulin stimulation.

MATERIALS AND METHODS

Materials

Anti-IRS-1 antibody and antirat p85 subunit of PI 3-kinase antibody were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Horseradish peroxidase (HRP)-conjugated monoclonal antiphosphotyrosine antibody (PY20H) was purchased from Transduction Laboratories, Inc. (Lexington, KY). LY294002, wortmannin, and rapamycin were obtained from Sigma (St. Louis, MO). PD98059 and phospho-specific and non-phospho-specific antibodies against Akt, p70 S6 kinase, and MAP kinase were obtained from New England Biolabs, Inc. (Beverly, MA). Monoclonal anti-GLUT4 antibody (1F8) was from East Acres Biologicals (Southbridge, MA). Antiinsulin receptor β-subunit antibody and HRP-conjugated antimouse and rabbit IgG antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Porcine insulin was kindly provided by the Lilly Research Laboratories (Indianapolis, IN). Lactacystin, MG132, and PSI were obtained from Calbiochem (La Jolla, CA). Polyvinylidene difluoride (PVDF) membrane was obtained from Millipore Corp. (Bedford, MA). DMEM and FCS were purchased from Life Technologies, Inc. (Gaithersburg, MD). Protein G-Sepharose was purchased from Pharmacia Biotech (Uppsala, Sweden). Electrophoresis reagents were from Bio-Rad Laboratories, Inc. (Hercules, CA). [1, 2, -3H] 2-Deoxyglucose was from NEN Life Science Products (Boston, MA). All other reagents and chemicals were from standard suppliers.

Cell Culture

3T3-L1 fibroblasts obtained from American Type Culture Collection (ATCC, Manassas, VA) were cultured in DMEM high glucose containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FCS in 10% CO2 atmosphere and induced to differentiate into adipocytes as described previously (48). 3T3-L1 adipocytes were used between 14 and 19 days after initiation of differentiation, when more than 95% of the cells exhibited an adipocyte-like phenotype.

Human embryonic kidney 293 cells obtained from ATCC were cultured in DMEM high glucose containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FCS in 5% CO2 atmosphere.

Infection of Recombinant Adenovirus Vectors

The recombinant adenoviruses, Ad5-p110CAAX containing bovine p110α cDNA with the CAAX motif at the COOH terminus and Ad5-CT that has no insert, were described previously (32). They were amplified in 293 cells and viral stock solutions with viral titer < 108 plaque forming units/ml were prepared. 3T3-L1 adipocytes were infected with the vectors by incubating the cells at indicated multiplicity of infection (m.o.i.) of viral stock solution in DMEM containing 2% heat-inactivated FCS overnight. The medium was replaced with DMEM containing 10% FCS, and the cells were used 48 h after infection.

Immunoprecipitation and Immunoblotting

Cells were solubilized at 4 C in an ice-cold buffer containing 20 mm Tris, pH 7.5, 140 mm NaCl, 1% Nonidet-P40, 1 mm EDTA, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 2 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 50 U/ml aprotinin, 1 μm leupeptin and 50 mm NaF, and centrifuged at 15,000 × g for 30 min to remove insoluble material. For immunoprecipitation of IRS-1, the cell lysates (500 μg protein) were incubated with anti-IRS-1 antibody (2 μg) for 16 h at 4 C, followed by incubation with protein G-Sepharose for 2 h. The cell lysates or immunoprecipitates were boiled in Laemmli buffer, resolved by SDS-PAGE with 7.5% or 12% acrylamide gels, and transferred to PVDF membranes. When antibody to phosphotyrosine was used, the membrane was blocked in TBS-T (10 mm Tris, pH 7.6, 150 mm NaCl, 0.1% Tween 20) containing 4% BSA overnight at 4 C and incubated with HRP-conjugated antiphosphotyrosine antibody (PY20H) for 1 h at room temperature. For immunoblotting with anti-IRS-1, p85, insulin receptor, or GLUT4 antibody, the membrane was blocked with TBS-T containing 5% nonfat dry milk and incubated with the indicated antibody for 1 h at room temperature, followed by incubation with HRP-conjugated secondary antibody. For detection of phospho- or non-phospho-Akt, p70 S6 kinase, or MAP kinase, the membrane was blocked with TBS-T containing 5% nonfat dry milk and incubated with the indicated antibody for 16 h at 4 C, followed by incubation with HRP-conjugated secondary antibody. The proteins were visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech).

2-DOG Uptake

Before the assay of [3H]2-DOG uptake, the cells were deprived of serum for 16 h and were stimulated with 20 nm insulin for the indicated period of time. Unlabeled 2-DOG and [3H]-2-DOG (0.1 mm , 0.74 kBq/well) were added to the cells in the KRP-HEPES buffer (10 mm HEPES, pH 7.4, 131.2 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 2.5 mm CaCl2, 2.5 mm NaH2PO4) with 1% BSA at 37 C and incubated for 4 min. Reaction was stopped by adding 10μ m cytochalasin B and washing cells with ice-cold PBS three times. The cells were solubilized in 1 ml of 0.2% SDS and 0.2 n NaOH. The radioactivity was quantitated in a liquid scintillation counter. The values of nonspecific uptake and absorption were determined by [3H]2-DOG uptake in the presence of 10 μm cytochalasin B, and the results were corrected for these values. Nonspecific uptake and absorption were always less than 10% of total uptake.

Statistical Analysis

Data were analyzed by Student’s t test. P values > 0.05 were considered significant.

Acknowledgement

This study was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (to T.H.) and by NIH Grant DK-33651 to (J.M.O.).

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