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Department of Anatomy and Physiology and Lipid Research Unit, Laval University Hospital Research Center, Ste-Foy, Québec, Canada G1V 4G2
Address all correspondence and requests for reprints to: Dr. André Marette, Department of Physiology and Lipid Research Unit, Laval University Hospital Research Center, 2705 Laurier boulevard, Ste-Foy, Québec, Canada G1V 4G2. E-mail: andre.marette{at}crchul.ulaval.ca.
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Abstract |
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5 min) and persistent increase in IRS-1-associated phosphatidylinositol 3-kinase activity and Akt phosphorylation. These results show that activation of the mTOR pathway is increased in liver and muscle of high fat-fed obese rats. In vitro studies with rapamycin suggest that mTOR/S6K1 overactivation contributes to elevated serine phosphorylation of IRS-1, leading to impaired insulin signaling to Akt in liver and muscle of this dietary model of obesity. |
Introduction |
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We (12) and others (13, 14, 15, 16) previously showed that chronic activation of the mTOR/S6K1 pathway by insulin, amino acids, or TNF-
promotes insulin resistance in fat and muscle cells through increased IRS-1 serine phosphorylation and degradation, leading to impaired PI3K stimulation. In fact, activation of the mTOR/S6K1 pathway by insulin and amino acids speeds up the temporal deactivation of PI3K activity even before IRS-1 degradation is detectable in L6 myocytes (12). It was thus proposed that the mTOR/S6K1 pathway represents a physiological feedback mechanism that negatively modulates the ability of insulin to transmit signal to PI3K via IRS-1. Whether overactivation of the mTOR/S6K1 pathway may contribute to defective activation of PI3K and Akt in insulin target tissues of obese insulin-resistant animals remains to be investigated. Sun and colleagues (17, 18) recently reported increased serine kinase activity in liver and muscle of obese insulin-resistant rodents, leading to increased inhibitory serine phosphorylation of IRS-1, a recognized molecular mechanism of insulin resistance for glucose metabolism (19). The identity of the serine kinase(s) responsible for elevated serine phosphorylation of IRS-1 remains to be determined, but the kinase activity was found to principally target serine(s) located in the middle region (amino acid 526–859 region) of the IRS-1 molecule (17, 18).
In the present study we tested whether mTOR/S6K1 overactivation may be involved in the pathogenesis of hepatic insulin resistance in diet-induced obesity. Our results indicate that mTOR and S6K1 activation are increased in liver and skeletal muscle of high fat-fed, obese rats, implicating these Ser/Thr kinases as potential mediators of insulin resistance in this model. We show, using the mTOR inhibitor rapamycin, that insulin activation of mTOR and S6K1 inhibits insulin signaling to the PI3K/Akt pathway in hepatic cells, possibly from increased inhibitory phosphorylation of IRS-1 on Ser636/Ser639.
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Materials and Methods |
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-32P]ATP, myo-[2-3H]inositol, protein A- and G-Sepharose, and antimouse or antirabbit immunoglobulin G conjugated to horseradish peroxidase were purchased from Amersham Pharmacia Biotech (Baie d’Urfé, Canada). Polyclonal antibodies against IRS-1 [raised against 20 C-terminal amino acids (C-20)], S6K1 (C-18), and Akt (C-20), S6K1 substrate, were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiphospho-specific antibodies against Akt (Ser473 and Thr308), IRS-1 (Ser636/Ser639), and S6K1 (Thr421/Ser424) were from New England Biolabs (Beverly, MA). Antibodies against IRS-1, IRS-2, p85/PI3K, and Akt substrate (Crosstide) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Human insulin was obtained from Eli Lilly & Co. (Toronto, Canada). Rapamycin was purchased from Biomol (Plymouth Meeting, PA). L--Phosphatidylinositol was obtained from Avanti Polar Lipids (Alabaster, AL). Oxalate-treated thin layer chromatography silica gel H plates were obtained from Analtech (Newark, DE). All other chemicals were of the highest analytical grade.
Cell culture and treatment
Human HepG2 hepatoma cells (gift from Dr. Luc Bélanger, Hotel Dieu Hospital, Laval University, Québec, Canada) were grown and maintained in monolayer culture in DMEM containing 10% (vol/vol) fetal bovine serum and 1% (vol/vol) antibiotic/antimycotic solution (10,000 U/ml penicillin, 10,000 µg/ml streptomycin, and 25 µg/ml amphotericin B) in an atmosphere of 5% CO2 at 37 C. Cells were deprived of serum 6 h before experimental treatments with or without rapamycin (25 nM) for 1 h and insulin (100 nM) for different times, as indicated in figure legends.
Animals
All experiments performed in this study were approved by the Laval University animal care and handling committee. Male Wistar rats (Charles River, Montréal, Canada), weighing 200–250 g at the beginning of the study, were housed individually in plastic cages in animal quarters maintained at 22 C with a 12-h dark, 12-h light schedule. Animals were fed either a low fat chow diet (Charles River rodent chow 5075, Ralston Purina, St. Louis, MO) or a purified high fat diet for 4 wk. The high fat diet was prepared in our laboratory as previously described (20). The composition of the high fat diet consisted of, as a percentage of total energy, 32.5% lard, 32.5% corn oil, 20% sucrose, and 15% protein, whereas the chow diet contained, as a percentage of total energy, 57.3% carbohydrate, 18.1% protein, and 4.5% fat. The high fat diet was supplemented with 1.4% vitamin mixture (Harlan Teklad, Madison, WI), 6.7% AIN-76 mineral mix, 0.2% choline bitartrate, and 0.004% butylated hydroxytoluene (ICN Nutritional Biochemicals, Cleveland, OH).
Whole body insulin sensitivity was determined in conscious overnight-fasted rats by performing an hyperinsulinemic-euglycemic clamp (insulin infused at 4 mU insulin/kg·min), as previously described (20). Other groups of chow and high fat-fed rats under ketamine/xylazine (87.5:12.5 mg/kg, ip) anesthesia were killed 5, 10, 15, 20, or 35 min after ip administration of insulin (0.075 U/g) or PBS as a control. In preliminary studies we determined that plasma insulin concentrations remain in the upper physiological range (>5 nM) throughout the 35-min period. Livers and extensor digitorum longus muscles were quickly excised and immediately frozen in liquid nitrogen.
Immunoprecipitation and immunoblotting
Protein extraction was performed in triplicate from livers, muscles, or HepG2 cells; extracts were prepared using buffer A (20 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 2 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin), and insoluble material was removed by centrifugation at 12,000 x g. Protein content was determined by the Lowry method using the DC reagent (Bio-Rad Laboratories, Inc., Richmond, CA). For direct Western blotting, protein extracts were resolved by SDS-PAGE. Where noted, proteins were immunoprecipitated (500 µg) after incubation with specific antibody (1–2 µg) or irrelevant immunoglobulin G for 18 h at 4 C. Immunocomplexes were collected, washed with buffer A three times, resuspended in Laemmli sample buffer, resolved in SDS-PAGE, and transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were blocked with 5% nonfat dry milk in 10 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween 20; incubated with primary antibody; and then reincubated with a secondary horseradish-conjugated antibody. Immunoreactive bands were visualized by enhanced chemiluminescence. Quantification was achieved by laser densitometry of the bands.
PI3K assay
PI3K activity was measured in IRS-1 and IRS-2 immunoprecipitates as previously described (12). In brief, after experimental treatment, medium was removed, and cells were rinsed twice in ice-cold PBS and lysed in 20 mM Tris (pH 7.4), 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% Nonidet P-40, 2 mM Na3VO4, and 10 mM NaF. Five hundred micrograms of lysates were immunoprecipitated with 2 µg anti-IRS-1 or anti-IRS-2 coupled to protein A-Sepharose overnight at 4 C. After washing, immune complexes were resuspended in 70 µl kinase buffer [8 mM Tris (pH 7.5), 80 mM NaCl, 0.8 mM EDTA, 15 mM MgCl2, 180 µM ATP, and 5 µCi [-32P]ATP] and 10 µl sonicated phosphatidylinositol mixture [20 µg L--phosphatidylinositol, 10 mM Tris (pH 7.5), and 1 mM EGTA] for 15 min at 30 C. Reaction was stopped by the addition of 20 µl 8 M HCl, mixed with 160 µl CHCl3/CH3OH (1:1), and centrifuged. The lower organic phase was spotted on an oxalate-treated silica gel thin layer chromatography plate and developed in CHCl3/CH3OH/H2O/NH4OH (60:47:11.6:2). The plate was dried and visualized by autoradiography with intensifying screen at –80 C.
Statistical analysis
Data are presented as the mean ± SE. The effects of the diet and rapamycin were analyzed by ANOVA. The effect of diet was analyzed by a 2 x 5 (standard diet/high fat, high sucrose (HFHS) diet and 0/5/10/20/35 min of insulin) factorial ANOVA. Likewise, the effect of rapamycin treatment was analyzed by a 2 x 4 (control/rapamycin and 0/5/15/30 min of insulin) factorial ANOVA. When justified by a significant treatment interaction, differences between individual group means were analyzed by Fisher’s protected least squares difference test. Significance was accepted at P < 0.05.
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Results |
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HFHS-fed obese rats and their lean chow-fed controls were studied in the basal fasting state or after injection of insulin at the times indicated in Figs. 1–3
. Protein extracts from livers of these animals were prepared and analyzed by Western blotting for the expression and phosphorylation levels of mTOR, S6K1, IRS-1, and Akt. mTOR and S6K1 phosphorylation on Ser2448 and Thr421/Ser424, respectively, was barely detectable in noninsulin-stimulated, chow-fed rats. Insulin injection rapidly increased the phosphorylation of both enzymes (t1/2, 10 min), reaching a 5-fold (mTOR) and approximately 20-fold (S6K1) phosphorylation at 35 min of insulin stimulation (Fig. 1, A–C). We next examined mTOR and S6K1 activation in liver of HFHS-fed obese insulin-resistant rats. Basal (noninsulin-stimulated) phosphorylation of both mTOR and S6K1 was markedly increased (4- to 5-fold) in liver of obese rats. Insulin-induced mTOR and S6K1 phosphorylation was also increased in the obese rats, but different patterns of activation were observed. Whereas mTOR phosphorylation in obese rat liver remained elevated compared with that in lean rats for up to 35 min of insulin stimulation, S6K1 phosphorylation was transiently increased at 5–10 min and then rapidly declined to basal levels within 35 min.
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, A and D, IRS-1 Ser636/Ser639 phosphorylation was low in liver of chow-fed, fasting rats. Insulin stimulation increased IRS-1 Ser636/Ser639 phosphorylation, peaking after 20–35 min of hormonal treatment, a time at which activation of both mTOR and S6K1 was prominent (Fig. 1, B and C). Basal IRS-1 Ser636/Ser639 phosphorylation was increased by about 2.5-fold in liver of obese rats compared with lean, chow-fed controls. Interestingly, insulin-induced IRS-1 Ser636/Ser639 phosphorylation was also increased, but only after 5 and 10 min of hormonal stimulation, suggesting that IRS-1 Ser636/Ser639 phosphorylation is accelerated in insulin-stimulated liver of obese, insulin-resistant rats. IRS-1 protein expression was not reduced by obesity or by this acute insulin treatment in liver of chow- or HFHS-fed rats. The time course of insulin-mediated IRS-1 Ser636/Ser639 phosphorylation was almost identical to that of S6K1 activation in liver of obese rats.
To evaluate the impact of increased activation of the mTOR pathway on insulin signaling to glucose metabolism, we next measured activation of Akt, a key effector of insulin-induced inhibition of glucose production (24) and of stimulation of glycogen synthesis (25). Basal Akt activation was slightly increased in liver of obese animals, and this reached the level of significance for the phosphorylation of Akt on Ser473. Insulin rapidly and markedly increased Akt phosphorylation on both Thr308 and Ser473 residues in liver of chow-fed rats (Fig. 2). In contrast, insulin’s ability to increase Akt phosphorylation was blunted in liver of obese rats, particularly at the 5- and 10-min points. Akt phosphorylation increased after 20–35 min of insulin injection in obese rats, but it remained lower than that of liver from insulin-injected, chow-fed rats. It is interesting to note that insulin-induced Akt activation was more severely reduced at the same time (i.e. 5–10 min) that S6K1 activation and IRS-1 Ser636/Ser639 phosphorylation were found to be markedly elevated in liver of obese rats.
We next tested whether activation of the mTOR/S6K1 pathway was also increased in skeletal muscle of HFHS-fed obese rats. As depicted in Fig. 3A and quantified in Fig. 3, B and C, basal (fasting state) phosphorylation of both mTOR and S6K1 was significantly increased (2- to 3-fold; P < 0.05) in extensor digitorum longus muscle of obese rats. Moreover, phosphorylation of mTOR and S6K1 was significantly enhanced in muscle of obese rats injected for only 5 min with insulin, compared with chow-fed animals. However, the insulin-dependent activation of both enzymes declined to basal levels within 15 min in HFHS-fed obese, but not chow-fed, rats. The inhibitory phosphorylation of IRS-1 on Ser636/Ser639 was increased in both the fasting state and 5 min after insulin injection in obese animals, but decreased to the basal level within 15 min of insulin stimulation (Fig. 3, A and D). Akt phosphorylation on Ser473 was also increased in obese fasted rats, but its activation by insulin was impaired both after 5 and 15 min of insulin stimulation (Fig. 3, A and E).
Insulin-induced mTOR/S6K1 activation causes IRS-1 Ser636/Ser639 phosphorylation and inhibition of the PI3K/Akt pathway in hepatocytes
The data obtained in HFHS- and chow-fed rats indicate that the mTOR pathway is overactivated in association with increased IRS-1 Ser636/Ser639 phosphorylation and impaired activation of Akt in liver and muscle of obese rats. Overactivation of the mTOR pathway may be explained by hyperinsulinemia, but several other humoral and local factors (e.g. TNF-) may be implicated. Moreover, these in vivo data do not establish the causal relationship between mTOR/S6K1 activation and impaired insulin signaling to Akt. To precisely address these issues, we next performed studies in cultured hepatocytes (HepG2 cells) exposed to insulin and in the presence of rapamycin, a specific inhibitor of the mTOR pathway.
HepG2 cells were preincubated for 1 h with the mTOR inhibitor rapamycin (25 nM) or vehicle (0.01% dimethylsulfoxide) before insulin stimulation. Cells were stimulated with 100 nM insulin for 5, 15, and 30 min or were left untreated. As shown in Fig. 4, A and C, mTOR phosphorylation on Ser2448 was increased by insulin, reaching maximal activation (4-fold) after 30 min of stimulation. A very similar pattern was observed for S6K1 phosphorylation on Ser424/Thr421, which was increased by 9-fold after 30 min of insulin stimulation (Fig. 4, A and D). Insulin also promoted IRS-1 phosphorylation on Ser636/Ser639 in HepG2 cells, as detected by direct immunoblotting of cell lysates (Fig. 4A) or by probing IRS-1 immunoprecipitates (Fig. 4B). The time dependence of insulin-induced IRS-1 phosphorylation on Ser636/Ser639 was very similar to that observed for activation of mTOR and S6K1 by the hormone (Fig. 4, compare C–E). As expected, the ability of insulin to activate mTOR and S6K1 was markedly reduced in cells preincubated with rapamycin (Fig. 4, A, C, and D), and this also blunted IRS-1 Ser636/Ser639 phosphorylation (Fig. 4E), indicating that activation of the mTOR pathway is responsible for phosphorylation of this site in insulin-treated hepatocytes.
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, A and B). Rapamycin pretreatment increased insulin-stimulated IRS-1-associated P13K and retarded its time-dependent deactivation. IRS-2-associated PI3K activity was also increased by insulin, but remained elevated throughout the 30-min period (Fig. 5, A and C). Rapamycin treatment also enhanced IRS-2-associated PI3K at 5 min, but not at 15 and 30 min. This suggests that inhibition of the mTOR pathway speeds up IRS-2-associated PI3K, but that this effect is transient.
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). Whereas insulin only transiently increased Thr308 phosphorylation (Fig. 5E), the hormone effect was maintained for up to 30 min on Ser473 (Fig. 5F). Rapamycin pretreatment increased insulin-stimulated Akt phosphorylation on both sites and slowed down the temporal dephosphorylation of Thr308. Thus, inhibition of the mTOR pathway by rapamycin increased insulin signaling to Akt in HepG2 cells. |
Discussion |
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(15), which is elevated in HFHS-fed obese rats (20). Finally, Ser2448 in mTOR can also be phosphorylated by amino acids (8, 27) through a mechanism independent from Akt activation, and this could also contribute to mTOR phosphorylation, because circulating amino acid levels are increased in obesity (28). Time-course experiments also revealed that mTOR and S6K1 are both rapidly phosphorylated in liver and muscle after insulin injection of obese animals, compared with lean chow-fed controls. In liver, S6K1 activation by insulin was only increased at early time points (5–10 min) despite the fact that mTOR-Ser2448 hyperphosphorylation was maintained for up to 35 min in obese animals. This apparent discrepancy between activation of mTOR and its downstream target S6K1 at the late time points of insulin injection may be explained by compensatory activation of Ser/Thr phosphatases that selectively target S6K1. Alternatively, it may indicate differential modulation of mTOR and S6K1 activity in obesity. Indeed, Ser2448 in mTOR can also be phosphorylated by glucagon and cAMP, but this is associated with inhibition of insulin-induced S6K1 activity in rat hepatocytes (26). Thus, glucagon and/or other cAMP-enhancing pathways may explain the persistent increase in mTOR-Ser2448 phosphorylation despite the time-dependent loss of S6K1 phosphorylation in liver of obese rats.
Our studies also strongly suggest that mTOR and/or S6K1 contribute to the pathogenesis of insulin resistance in liver and muscle tissues. Increased phosphorylation of IRS-1 on serine residues is considered a molecular hallmark of insulin resistance in insulin target tissues (19). In vitro studies suggest that both mTOR and S6K1 can phosphorylate multiple serines in IRS-1 (12, 14, 15, 16, 29, 30, 31, 32). More recently, Gual et al. (21) confirmed that Ser636/Ser639 is a target of the mTOR pathway in vivo, because rapamycin treatment fully blocked phosphorylation of this site in muscle and fat of normal mice. However, the contribution of the mTOR pathway to phosphorylation of Ser636/Ser639 in IRS-1 remains to be established in obese insulin-resistant animals. We found that in liver of obese rats, elevated IRS-1 phosphorylation on Ser636/Ser639 and impaired Akt activation by insulin were evident at early time points, suggesting that S6K1, and not mTOR directly, may be more linked with hepatic insulin resistance in obesity. In muscle, however, the time course of overactivation of mTOR and S6K1 were similar and paralleled the elevated IRS-1 Ser636/Ser639 phosphorylation in HFHS-fed obese rats. The marked decline in S6K1 activity in muscle of obese rats 15 min after insulin injection is in agreement with a previous study showing a lack of insulin activation of S6K1 in skeletal muscle of gold thioglucose-treated, obese mice compared with lean mice (33). Indeed, S6K1 activity was measured after 15 min of insulin stimulation in the latter study, a time at which overactivation of S6K1 is no longer apparent in the obese rat model. Importantly, a role for S6K1 as a primary inducer of inhibitory serine phosphorylation of IRS-1 and insulin resistance in obesity is also supported by the recent observation that S6K1 knockout mice are more resistant to diet-induced obesity and more insulin sensitive than wild-type mice (34).
Our data for liver of obese rats suggest a role for the overactivation of the mTOR pathway in the development of insulin resistance in liver of obese rats. However, the connection between the mTOR pathway and the phosphorylation of Ser636/Ser639 in IRS-1 in obese animals is correlative, because multiple kinases may increase phosphorylation of this site (see below). In contrast, such a connection is supported by our in vitro studies in HepG2 cells, showing that inhibition of mTOR/S6K1 by rapamycin reverses insulin-mediated IRS-1 phosphorylation on Ser636/Ser639 while increasing Akt activation by insulin. Rapamycin is a very selective inhibitor of the mTOR pathway, with no inhibitory activity toward other known kinases even at concentrations in the high nanomolar range (35). These in vitro studies also suggest that hyperinsulinemia contributes to mTOR/S6K1 overactivation in the fasting state and probably in the postprandial state, because glucose-induced insulin secretion is increased by up to 2-fold in HFHS-fed, obese rats compared with chow-fed controls (Dombrowski, L., and A. Marette, unpublished observations). These studies in liver cells are in line with recent studies by Gual et al. (21), showing that rapamycin blunted phosphorylation of IRS-1 on Ser632 (Ser636 in humans) by prolonged insulin treatment of 3T3-L1 adipocytes as well as fat and muscle tissues of normal mice in vivo.
Although evidence is accumulating about the potential role of the mTOR/S6K1 pathway in promoting inhibitory serine phosphorylation of IRS-1 and insulin resistance in obesity, other pathways are also likely to contribute to this pathogenic mechanism. Thus, insulin has been recently shown to acutely promote phosphorylation of Ser612 and Ser632/Ser636 through a MAPK-dependent pathway in 3T3-L1 adipocytes (21). Moreover, cultured muscle cells from type 2 diabetic subjects have increased MAPK (extracellular signal regulated kinase-1 and -2) activity and the MAPK inhibitor PD98059 blunted IRS-1 phosphorylation on Ser636 in these cells (29). Interestingly, both Ser612 and Ser632/Ser636 phosphorylation by insulin were found to be acutely dependent on MAPK (within minutes), but were mostly linked to mTOR activation upon prolonged insulin treatment (1 h) (21) in normal lean mice. Thus, our finding of an accelerated activation of mTOR and S6K1 phosphorylation by insulin in obese animals suggest that this pathway may be more rapidly promoting Ser636/Ser639 phosphorylation in obese animals. Other kinases link to inflammatory pathways are also thought to be responsible for IRS-1 serine phosphorylation in obesity. Thus, Ser307 in IRS-1 has been reported to be targeted by the c-Jun N-terminal kinase (JNK) and inhibitor 
B kinase (36, 37, 38), which are both increased in obesity (38, 39). Interestingly, an unidentified kinase was recently shown to phosphorylate Ser789 in IRS-1 in liver of obese rats (18). It will be interesting to see whether this kinase, which is neither JNK nor extracellular signal-regulated kinase, turns out to be mTOR or S6K1, which we show here to be overactivated in liver of obese rats.
In summary, this study shows that the activation of mTOR and S6K1 is increased in liver and skeletal muscle of high fat-fed, obese rats. We demonstrate, using rapamycin in vitro, that mTOR/S6K1 activation by insulin increases Ser636/Ser639 phosphorylation of IRS-1, leading to impaired PI3K/Akt activation in liver cells. Taken together, these results strongly suggest that mTOR and/or S6K1 are potential mediators of elevated inhibitory phosphorylation of IRS-1 on Ser636/Ser639 and tissue insulin resistance in obesity.
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Acknowledgments |
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Footnotes |
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First Published Online December 16, 2004
Abbreviations: HFHS, High fat, high sucrose; JNK, c-Jun N-terminal kinase; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; PVDF, polyvinylidene difluoride.
Received July 16, 2004.
Accepted for publication December 6, 2004.
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in vitro. J Biol Chem 274:34493–34498B kinase ß. Science 293:1673–1677This article has been cited by other articles:
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) and insulin-induced (
) phosphorylation of mTOR, S6K1, and IRS-1 are shown. Results are expressed in this and subsequent figures as the mean ± SEM. *, P < 0.05; **, P < 0.01 (vs. chow-fed rats at same time point).
