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Endocrinology, doi:10.1210/en.2003-1574
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Endocrinology Vol. 145, No. 7 3215-3223
Copyright © 2004 by The Endocrine Society

Impact of Src Homology 2-Containing Inositol 5'-Phosphatase 2 on the Regulation of Insulin Signaling Leading to Protein Synthesis in 3T3-L1 Adipocytes Cultured with Excess Amino Acids

Shihou Murakami, Toshiyasu Sasaoka, Tsutomu Wada, Kazuhito Fukui, Kiyofumi Nagira, Hajime Ishihara, Isao Usui and Masashi Kobayashi

First Department of Internal Medicine (S.M., T.W., K.F., K.N., I.U., M.K.) and Department of Clinical Pharmacology (T.S.), Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan; and Sainou Hospital (H.I.), Toyama 930-0887, Japan

Address all correspondence and requests for reprints to: Toshiyasu Sasaoka, M.D., Ph.D., Department of Clinical Pharmacology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: tsasaoka-tym{at}umin.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) possesses 5'-phosphatase activity to specifically hydrolyze the phosphatidylinositol 3-kinase product PI(3,4,5)P3 in the regulation of insulin signaling. In the present study, we examined the impact of SHIP2 on the regulation of insulin signaling leading to protein synthesis in 3T3-L1 adipocytes cultured with standard and excess concentrations of amino acids. Insulin-induced translocation of PDK1 to the plasma membrane, phosphorylation of Akt and p70S6-kinase and ribosomal protein S6, increase in the amount of 4E-BP1 {gamma}-form, association of eIF4E with eIF4G, and protein synthesis were decreased by overexpression of wild-type SHIP2 by adenovirus-mediated gene transfer. The effect of SHIP2 overexpression on the regulation of insulin-induced phosphorylation of Akt and p70S6-kinase was somewhat augmented by the incubation with 5-fold excess concentrations of amino acids for 30 min. In contrast, the impact of SHIP2 expression was diminished in insulin-induced phosphorylation of p70S6-kinase and S6, but not of Akt, after the incubation for 16 h. Interestingly, incubation with the excess concentrations of amino acids for 30 min induced activation of phosphatidylinositol 3-kinase and phosphorylation of Akt, whereas phosphorylation of p70S6-kinase and S6 was decreased. Furthermore, although the exposure for longer time periods up to 24 h did not elicit phosphorylation of Akt, it markedly induced phosphorylation of p70S6-kinase and S6. These results indicate that SHIP2 plays an important role in the negative regulation of insulin signaling for the protein synthesis and that the impact of SHIP2 is altered, dependent on the acute or chronic exposure of excess concentrations of amino acids in culture.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN INDUCES TYROSINE phosphorylation of the insulin receptor substrate (IRS) family of proteins on the tyrosine residues (1, 2, 3, 4, 5). IRS proteins propagate insulin signals to the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, resulting in the activation of the p110 catalytic subunit (1, 2, 3, 4, 5). Activation of the PI3-kinase is important for exerting various metabolic actions of insulin including glucose uptake, glycogen synthesis, antilipolysis, and protein synthesis (1, 2, 3, 4, 5). PI3-kinase functions as a lipid kinase to produce PI(3,4,5)P3 from PI (4, 5)P2. PI(3,4,5)P3 is thought to act as a key lipid second messenger to further downstream molecules for the metabolic action of insulin (6, 7). We and others (8, 9) identified as Src homology 2-containing inositol 5'-phoshatase 2 (SHIP2) possessing 5'-phosphatase activity to specifically hydrolyze PI3-kinase product PI(3,4,5)P3 to PI(3,4)P2. Subsequent reports showed that overexpression of SHIP2 inhibited insulin-induced glucose uptake and glycogen synthesis via its 5'-phosphatase activity in 3T3-L1 adipocytes and L6 myocytes (10, 11). Targeted disruption of the SHIP2 gene in mice showed increased insulin sensitivity without affecting biological systems other than insulin signaling (12). These findings indicate that SHIP2 is a physiologically important negative regulator relatively specific to insulin signaling apparently for the glucose uptake and glycogen synthesis. In addition, insulin also stimulates protein synthesis as another important metabolic action (3, 4, 5). However, it is unknown whether SHIP2 is in fact involved in the regulation of insulin-induced protein synthesis.

Oral food intake causes acute rise in plasma concentrations of amino acids, which are essential and indispensable for life (13). Amino acids not only are the simple substrate for the protein synthesis but also function as the nutrient signal molecules in the protein synthesis (14). The rapamycin-sensitive pathway via mammalian target of rapamycin (mTOR) is known to be a target activated by the nutrient, which controls the mammalian translation machinery via activation of p70 S6 kinase (S6K1) and inhibition of the eukaryotic initiation factor (eIF)4E inhibitor, 4E-BP1 (also known as PHAS-I) (15, 16, 17, 18). In addition to the physiological role of amino acids, previous studies have shown that fasting plasma amino acid concentrations are elevated both in rodents and human subjects with obesity (19, 20). Thus, the chronic elevation of amino acid concentrations appears to be associated with the pathological state including the insulin resistance syndrome (19). These facts prompted us to examine the impact of SHIP2 on insulin signaling leading to protein synthesis in the state of different nutrient concentrations. Thus, it would be important to clarify whether the control of insulin signaling via SHIP2 is regulated by the state of nutrition.

In the present study, we investigated the role of SHIP2 in insulin signaling leading to the protein synthesis by overexpressing wild-type SHIP2 using adenovirus-mediated gene transfer in 3T3-L1 adipocytes. The effect of SHIP2 on insulin-induced subcellular redistribution of phosphoinositide-dependent kinase 1 (PDK1), phosphorylation of Akt and S6K1 and ribosomal protein S6 (rp S6), increase in the amount of 4E-BP1 {gamma}-form, association of eIF4E with eIF4G, and protein synthesis was studied. Furthermore, the impact of SHIP2 in the different nutritional state on the regulation of insulin signaling leading to the protein synthesis was examined in the cell culture with excess concentrations of amino acids for various time periods.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human crystal insulin was provided by Novo Nordisk Pharmaceutical Co., (Copenhagen, Denmark). [{gamma}-32P]ATP (111 TBq/mmol) was purchased from NEN Life Science Products, Inc. (Boston, MA). Redivue Pro-mix L-[35S] in vitro cell labeling mix (>37 TBq/mmol) was from Amersham Biosciences Corp. (Piscataway, NJ). A polyclonal anti-SHIP2 antibody was described previously (9, 10). A monoclonal antiphosphotyrosine antibody (PY20) was from Transduction Laboratories (Lexington, KY). A polyclonal anti-IRS-1 antibody and a polyclonal anti-platelet-derived growth factor (PDGF) ß-receptor antibody were from Upstate Biotechnology (Lake Placid, NY). A polyclonal anti-Ser473 phospho-specific Akt antibody, a polyclonal anti-phospho-specific p70 S6K1 antibody, a polyclonal anti-phospho-specific rp S6 antibody, a polyclonal anti-S6K1 antibody, a polyclonal anti-rp S6 antibody, and a polyclonal anti-eIF4E antibody were from Cell Signaling Technology, Inc. (Beverly, MA). A polyclonal antiinsulin receptor antibody, a polyclonal anti-PDK1 antibody, a polyclonal anti-Akt antibody, a polyclonal anti-4E-BP1 antibody, and a polyclonal anti-eIF4G antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence reagents were from Amersham Pharmacia Biotech Corp. (Uppsala, Sweden). DMEM and MEM amino acid solutions were from Life Technologies, Inc. Japan (Tokyo). All other reagents were of analytical grade and purchased from Sigma Chemical Co. (St. Louis, MO) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Cell culture and infection of adenovirus
3T3-L1 fibroblasts were grown and passaged in DMEM supplemented with 10% newborn calf serum. Cells at 2–3 d post confluence were used for differentiation. The differentiation medium contained 10% fetal calf serum (FCS), 250 nM dexamethasone, 0.5 mM isobutyl methylxanthine, and 500 nM insulin. After 3 d, the differentiation medium was replaced with postdifferentiation medium containing 10% FCS and 500 nM insulin. After another 3 d, postdifferentiation medium was replaced with DMEM supplemented with 10% FCS. Adenovirus vectors encoding wild-type (WT)-SHIP2, constitutively active form of rat Akt1 by adding the Src myristration signal at the N terminus (Myr-Akt), and ß-galactosidase (LacZ) were described previously (10). WT-SHIP2 and Myr-Akt were transiently expressed in differentiated 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer (10). A multiplicity of infection (m.o.i.) of 40 pfu/cell was used to infect 3T3-L1 adipocytes in DMEM containing 2% FCS, with the virus being left on the cells for 16 h before removal. Subsequent experiments were conducted 24–48 h after initial addition of the virus. The efficiency of adenovirus-mediated gene transfer of WT-SHIP2 and Myr-Akt was approximately 90%.

Subcellular fractionation
The cells were washed twice with PBS and once with HES buffer [255 mM sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 2 µg/ml aprotinin, and 50 ng/ml okadaic acid (pH 7.4)] and immediately homogenized by 20 strokes with a motor-driven homogenizer in HES buffer at 4 C. The homogenates (two 10-cm-diameter dishes per condition) were subjected to subcellular fractionation as described previously to isolate plasma membrane (PM) and cytosol (17). In brief, the homogenates were centrifuged at 19,000 x g for 20 min. The resulting supernatant was centrifuged at 250,000 x g for 90 min. The remaining supernatant was concentrated by Centricon-30 (Amicon Inc., Beverly, MA) and used as cytosol. The pellet obtained from the initial spin was resuspended in HES buffer, layered onto a 1.12 M sucrose cushion, and centrifuged at 100,000 x g in a swing rotor for 60 min. A white fluffy band at the interface was collected and resuspended in HES buffer and centrifuged at 40,000 x g for 20 min, yielding a pellet of PM. The samples were adjusted to a final protein concentration of 1–3 mg/ml, which was measured by the Bradford method, and stored at –80 C until use.

Amino acid treatment
MEM containing 5-fold concentrations of amino acids (X5) were prepared by mixing the solutions containing 4-fold concentration of amino acids in the regular MEM. The amino acids and their concentrations in the standard MEM (X1) are as follows: arginine, 600 µM; cystine, 100 µM; glutamine, 2 mM; histidine, 200 µM; isoleucine, 400 µM; leucine, 400 µM; lysine, 397 µM; methionine, 101 µM; phenylalanine, 194 µM; threonine, 403 µM; tryptophan, 49 µM; tyrosine, 199 µM; and valine, 393 µM.

Immunoprecipitation and Western blotting
The cells or the plasma membrane preparation was lysed in a buffer containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium deoxycholate, 1 mM ß-glycerophosphate, 1% Triton X-100, 1 mM PMSF, 1 mM Na3VO4, 50 mM sodium fluoride, 10 µg/ml aprotinin, and 10 µM leupeptin (pH 7.4) for 15 min at 4 C. The lysates were centrifuged to remove insoluble materials. The supernatants (100 µg protein) were immunoprecipitated with antibodies for 2 h at 4 C. The precipitates or the lysates were then separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes using a Transblot apparatus (Bio-Rad Laboratories, Hercules, CA) (10, 18). The membranes were blocked in a buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 2.5% BSA or 5% nonfat milk (pH 7.5) for 2 h at 20 C. The membranes were then probed with antibodies for 2 h at 20 C or 16 h at 4 C. After the membranes were washed in a buffer containing 50 mM Tris, 150 mM NaCl, and 0.1% Tween 20 (pH 7.5), blots were incubated with a horseradish peroxidase-linked second antibody and subjected to enhanced chemiluminescence detection using ECL reagent according to the manufacturer’s instructions (Amersham).

Measurement of protein synthesis
Total protein synthesis was measured by the incorporation of [35S]methionine and [35S]cysteine into proteins. In brief, cells were incubated in the methionine and cysteine-free DMEM for 3 h and treated with insulin for 1 h. After the 35S-labeled methionine and cysteine were added for 1 h, the cells were washed twice with ice-cold 0.9% NaCl. The cells were solubilized in a buffer containing 1% Triton X-100 and 1% deoxycholate. Aliquots of the cell lysates were spotted onto the filter paper (Whatman, Kent, UK). The filter papers were washed once with ice-cold 5% trichloroacetic acid (TCA), once with boiling 5% TCA, twice with ice-cold TCA, and once with 100% ethanol. The washed filter papers were dried, and radiolabeled amino acids incorporated into proteins were measured by liquid scintillation counting (21).

Measurement of PI3-kinase activity
Serum-starved 3T3-L1 adipocytes grown in 10-cm dishes were stimulated with 17 nM insulin at 37 C for 10 min. The cells were lysed in a buffer containing 20 mM Tris, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.1 mM Na3VO4, 1% Nonidet P-40, 10% glycerol, 2 mM PMSF, and 10 µg/ml aprotinin (pH 7.6). The cell lysates were centrifuged to remove insoluble materials. The supernatants were immunoprecipitated with antiphosphotyrosine antibody for 2 h at 4 C. The precipitates were washed twice with buffer A [Tris-buffered saline, 1% Nonidet P-40, 0.1 mM Na3VO4, and 1 mM dithiothreitol (DTT) (pH 7.6)], twice with buffer B [100 mM Tris, 500 mM LiCl, 0.1 mM Na3VO4, and 1 mM DTT (pH 7.6)], and twice with buffer C [10 mM Tris, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT (pH 7.6)]. The phosphorylation reaction was started by adding 20 µl PtdIns solution containing 0.5 mg/ml PtdIns, 50 mM HEPES, 1 mM NaH2PO4, and 1 mM EGTA (pH 7.6) at 20 C, followed by addition of 10 µl of the reaction mixture containing 250 µM [{gamma}-32P]ATP (0.37 Mbq/tube), 100 mM HEPES, and 50 mM MgCl2 (pH 7.6) for 5 min. The reaction was stopped by the addition of 15 µl of 8 M HCl. The products were extracted by adding 130 mM chloroform/methanol (1:1) followed by centrifugation. The organic phase was removed and spotted on Silica Gel thin-layer chromatography plate (Merck, Whitehouse Station, NJ). The plates were developed and dried (10). The phosphorylated inositol was visualized by autoradiography and quantitated by the BAS 2000 image analyzer (Fuji Film, Tokyo, Japan).

Statistical analysis
The data are represented as the mean ± SE. P values were determined by Student t test, and P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of SHIP2 overexpression on insulin-induced subcellular localization of PDK1.
SHIP2 possesses 5'-phosphatase activity hydrolyzing PI3-kinase product PI(3,4,5)P3 to PI(3,4)P2 in 3T3-L1 adipocytes (10). PDK1 is a direct target molecule of PI3-kinase, important for mediating the various actions of insulin including protein synthesis (22, 23). Although insulin treatment does not alter the total activity of PDK1, it induces a subcellular redistribution of PDK1 to the plasma membrane for the functioning (24). Therefore, we examined the effect of SHIP2 overexpression on insulin-induced subcellular redistribution of PDK1 to the plasma membrane. As shown in Fig. 1Go, insulin treatment increased the amount of PDK1 in the plasma membrane fraction. The amount PDK1 in the cytosol was not significantly altered in response to insulin, possibly because a small part of PDK1 in the cytosol translocated to the plasma membrane. Overexpression of SHIP2 decreased the amount of PDK1 in the plasma membrane fraction by 67.0 ± 4.5% in 3T3-L1 adipocytes. To assure equal amounts of protein were loaded among the samples, the PM fraction was immunoblotted with anti-PDGF receptor antibody (Fig. 1CGo). Five-fold greater expression of SHIP2 than the levels of endogenous SHIP2 was confirmed by immunoblotting of the cell lysates with anti-SHIP2 antibody (Fig. 1DGo).



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  		FIG. 1. Effect of SHIP2 overexpression on insulin-induced subcellular localization of PDK1. 3T3-L1 adipocytes were transfected with either LacZ or WT-SHIP2 at an m.o.i. of 40 pfu/cell. The cells were serum starved for 16 h and then treated with 17 nM insulin for 10 min. The cells were homogenized and subjected to subcellular fractionation to yield the PM and cytosol fractions. Samples in the PM (A) and cytosol (B) fractions were separated by SDS-PAGE and immunoblotted with anti-PDK1 antibody. The amount of PDK1 at the PM and cytosol corrected for the loaded protein amount was quantitated by densitometry. Results are the mean ± SE of three separate experiments. *, P < 0.05 vs. amounts of PDK1 in LacZ-transfected cells. C, Samples in the PM fraction were separated by SDS-PAGE and immunoblotted with anti-PDGF receptor antibody. D, The total cell lysates were separated by SDS-PAGE and immunoblotted with anti-SHIP2 antibody.

 
Effect of SHIP2 overexpression on insulin-induced phosphorylation of Akt, S6K1, and rp S6
Akt, S6K1, and rp S6 are located downstream of PI3-kinase and PDK1 and are involved in the signaling cascade of insulin-induced protein synthesis (2, 3, 25, 26, 27, 28). Because the activity of Akt, S6K1, and rp S6 is regulated by their phosphorylations, we examined the effect of SHIP2 expression on insulin-induced phosphorylation of Akt, S6K1, and rp S6 (Fig. 2AGo). Insulin treatment-induced phosphorylation of Akt, S6K1, and rp S6 in a dose-dependent manner. Overexpression of SHIP2 inhibited insulin-induced phosphorylation of Akt, S6K1, and rp S6. At 1.7 nM insulin concentration, insulin-induced phosphorylation of Akt, S6K1, and rp S6 was inhibited by 43.4 ± 2.9, 33.3 ± 3.1, and 27.5 ± 1.8%, respectively, by overexpression of WT-SHIP2 (Fig. 2BGo). The equal amount of protein loaded among the samples was confirmed by immunoblotting the cell lysates with anti-Akt, anti-S6K1, and anti-rp S6 antibodies. In addition, 5-fold greater amount SHIP2 expression, compared with the endogenous SHIP2, was confirmed by immunoblotting with anti-SHIP2 antibody. Because insulin-induced phosphorylation of S6K1 and rp S6 are downstream events of PI3-kinase, and not of Akt (2, 3, 25, 26, 27), coexpression of constitutively active form of Akt (Myr-Akt) did not affect the SHIP2 inhibition of insulin-induced phosphorylation of S6K1 and rp S6 (data not shown).



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  		FIG. 2. Effect of SHIP2 overexpression on insulin-induced phosphorylation of Akt, S6K1, and rp S6. 3T3-L1 adipocytes were transfected with either LacZ or WT-SHIP2 at an m.o.i. of 40 pfu/cell. The cells were serum starved for 16 h and then treated with various concentrations of insulin for 10 min. A, The cell lysates were separated by SDS-PAGE and immunoblotted with the specified antibody. B, The amount of phosphorylated Akt, S6K1, and rp S6 corrected for the loaded protein amount was quantitated by densitometry. Results are the mean ± SE of three separate experiments. *, P < 0.05 vs. amounts of Akt, S6K1, and rp S6 at respective concentrations of insulin in LacZ-transfected cells.

 
Effect of SHIP2 overexpression on insulin-induced increase in the amount of 4E-BP1 {gamma}-form and association of eIF4E with eIF4G
4E-BP1 is implicated in the translation initiation of mRNA (16, 26, 27). 4E-BP1 is composed of {alpha}-, ß-, and {gamma}-forms based on the mobility shift of 4E-BP1. Both {alpha}- and ß-forms of 4E-BP1 are mainly seen in the basal state, and insulin increases the amount of {gamma}-form for the protein synthesis (16, 18, 28, 29, 30). In fact, insulin treatment increased the amount of 4E-BP1 {gamma}-form in a dose-dependent manner. In accordance with the results of S6K1 and rp S6 phosphorylations, overexpression of SHIP2 inhibited insulin-induced increase in the amount of 4E-BP1 {gamma}-form. At 1.7 nM insulin concentration, the amount of 4E-BP1 {gamma}-form was decreased by 42.5 ± 3.1% by overexpression of SHIP2 (Fig. 3AGo). After phosphorylation of 4E-BP1, it dissociates from eIF4E, resulting in association of eIF4E with eIF4G in the signaling leading to protein synthesis (15, 16, 31, 32). We further examined the effect of SHIP2 expression on insulin-induced association of eIF4E with eIF4G. Consistent with the results of 4E-BP1 studies, insulin-induced association of eIF4E with eIF4G was inhibited by overexpression of SHIP2. At 1.7 nM insulin concentration, the amount of eIF4E associated with eIF4G was decreased by 48.6 ± 2.1% by overexpression of SHIP2 (Fig. 3BGo). In contrast to the results with S6K1 and rp S6, because insulin-induced increase in the amount of 4E-BP1 {gamma}-form and association of eIF4E with eIF4G are downstream events of Akt activation (28, 29, 30, 31, 32), coexpression of Myr-Akt rescued the effect of SHIP2 overexpression on insulin-induced increase in the amount of 4E-BP1 {gamma}-form and association of eIF4E with eIF4G (data not shown).



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  		FIG. 3. Effect of SHIP2 overexpression on insulin-induced increase in the amount of 4E-BP1 {gamma}-form and association of eIF4E with eIF4G. 3T3-L1 adipocytes were transfected with either LacZ or WT-SHIP2 at an m.o.i. of 40 pfu/cell. The cells were serum starved for 16 h and then treated with various concentration of insulin for 10 min. A, The cell lysates were separated by SDS-PAGE and immunoblotted with anti-4E-BP1 antibody. The amount of 4E-BP1 {gamma}-form was quantitated by densitometry. Results are the mean ± SE of three separate experiments. *, P < 0.05 vs. amounts of 4E-BP1 {gamma}-form at respective concentrations of insulin in LacZ-transfected cells. B, The cell lysates were immunoprecipitated with anti-eIF4G antibody. The precipitates were separated by SDS-PAGE and immunoblotted with anti-eIF4E antibody. Results are the mean ± SE of three separate experiments. *, P < 0.05 vs. amounts of eIF4E at respective concentrations of insulin in LacZ-transfected cells.

 
Effect of SHIP2 overexpression on insulin-induced protein synthesis
Because insulin signalings leading to protein synthesis were effectively inhibited by overexpression of SHIP2, we directly investigated the effect of SHIP2 overexpression on insulin-induced protein synthesis. As shown in Fig. 4Go, insulin stimulated 35S-methionine and 35S-cysteine incorporation into proteins by 105.4 ± 15.2%. Overexpression of SHIP2 significantly inhibited insulin-induced protein synthesis by 48.4 ± 9.8% in 3T3-L1 adipocytes.



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  		FIG. 4. Effect of SHIP2 overexpression on insulin-induced protein synthesis. 3T3-L1 adipocytes were transfected with either LacZ or WT-SHIP2 at an m.o.i. of 40 pfu/cell. The cells were incubated in methionine and cysteine-free DMEM for 3 h and treated with insulin for 1 h. Then, incorporation of [35S]methionine and [35S]cysteine into protein for 1 h was measured. Results are the mean ± SE of three separate experiments. *, P < 0.05 vs. protein synthesis in insulin-stimulated LacZ-transfected cells.

 
Effect of SHIP2 overexpression on insulin-induced phosphorylation of Akt, S6K1, and rp S6 in excess amino acids
The degree of protein synthesis can be regulated by the state of nutrition including the concentration of amino acids contained in the culture media (33). Therefore, we examined the effect of SHIP2 expression on insulin-induced phosphorylation of Akt, S6K1, and rp S6 in both standard and excess amino acids for different time periods (Fig. 5Go). Interestingly, the decrease in insulin-induced phosphorylation of Akt by expression of SHIP2 was somewhat further augmented in the exposure of 5-fold concentrations of amino acids for 30 min, whereas the degree of inhibition in the cell culture with the excess amino acids for 16 h was similar to that with standard concentrations of amino acids. Furthermore, the degree of insulin-induced phosphorylation of S6K1 and rp S6 was decreased in the cell culture with the excess amino acids for 30 min, compared with that with standard concentrations of amino acids. Regardless of the alteration, overexpression of SHIP2 effectively inhibited insulin-induced phosphorylation of S6K1 and rp S6. Insulin induced phosphorylation of S6K1 and rp S6 in the cell culture with 5-fold concentration of amino acids for 16 h to the similar extent seen in the cell culture with standard concentrations of amino acids. In contrast to the results in the cell culture with standard concentrations of amino acids and with the excess amino acids for 30 min, the effect of SHIP2 overexpression on insulin-induced phosphorylation of S6K1 and rp S6, and not Akt, was diminished in the cell culture with 5-fold concentrations of amino acids for 16 h.



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  		FIG. 5. Effect of SHIP2 overexpression on insulin-induced phosphorylation of Akt, S6K1, and rp S6 in excess amino acids. 3T3-L1 adipocytes were transfected with either LacZ or WT-SHIP2 at an m.o.i. of 40 pfu/cell. Serum-starved transfected cells were incubated with standard concentrations of amino acids (X1) or with 5-fold excess of amino acids (X5) for 30 min or 16 h, and the cells were stimulated with 17 nM insulin for 10 min. A, The cell lysates were separated by SDS-PAGE and immunoblotted with the specified antibody. B, The amount of phosphorylated Akt, p70S6-kinase, and S6 corrected for the loaded protein amount was quantitated by densitometry. Results are the mean ± SE of three separate experiments.

 
Effect of SHIP2 overexpression on insulin-induced tyrosine phosphorylation of insulin receptor and IRS-1 and activation of PI3-kinase in excess amino acids
Because the impact of SHIP2 on the regulation of insulin-induced phosphorylation of Akt, S6K1, and rp S6 was altered, dependent on the cell culture media with the excess amino acids for 30 min or 16 h, we investigated the possible molecular mechanisms by which treatment with high concentrations of amino acids causes the change in the regulation of these phosphorylations. Overexpression of SHIP2 did not affect insulin-induced tyrosine phosphorylation of insulin receptor and IRS-1 in the cell culture with standard concentrations of amino acids, 5-fold concentrations of amino acids for 30 min, or 5-fold concentrations of amino acids for 16 h (Fig. 6Go, A and B). In addition, overexpression of SHIP2 affected neither insulin-induced activation of PI3-kinase in the cell culture with standard concentrations of amino acids, with the excess amino acids for 30 min, nor the excess amino acids for 16 h. However, treatment of the cells with 5-fold concentrations of amino acids alone for 30 min, but not 16 h, stimulated PI3-kinase activity in 3T3-L1 adipocytes (Fig. 6CGo).



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  		FIG. 6. Effect of SHIP2 overexpression on insulin-induced tyrosine phosphorylation of insulin receptor and IRS-1 and activation of PI3-kinase in excess amino acids. 3T3-L1 adipocytes were transfected with either LacZ or WT-SHIP2 at an m.o.i. of 40 pfu/cell. Serum-starved transfected cells were incubated with standard concentrations of amino acids (X1) or with 5-fold excess of amino acids (X5) for 30 min or 16 h, and the cells were stimulated with 17 nM insulin for 10 min. The cell lysates were immunoprecipitated with antiinsulin receptor antibody (A) or anti-IRS-1 antibody (B), and then the precipitates were immunoblotted with phosphotyrosine antibody. C, The cell lysates were immunoprecipitated with antiphosphotyrosine antibody. The washed immunoprecipitates were assayed for PI3-kinase activity with phosphatidylinositol as a substrate, and the labeled phosphatidylinositol (3 ) phosphate product (PIP3) was resolved by thin-layer chromatography and quantitated by BAS 2000. Results are the mean ± SE of three separate experiments.

 
Time course of excess amino acid-induced phosphorylation of Akt, S6K1, and rp S6
Recent studies indicated that phosphorylation of S6K1 and rp S6 occurs independent of insulin treatment, which is dependent on the availability of amino acids (34). Therefore, we examined the time course of 5-fold concentrations of amino acid-induced phosphorylation of Akt (Fig. 7AGo), S6K1 (Fig. 7BGo), and rp S6 (Fig. 7CGo). In accordance with the results of PI3-kinase activity, the amino acid excess alone induced transient phosphorylation of Akt at 30 min, declining thereafter. Considerable amounts of S6K1 and rp S6 were phosphorylated, even in the basal states. Interestingly, the degree of the phosphorylation of both S6K1 and rp S6 was decreased by the exposure of 5-fold concentrations of amino acids for short periods of time up to 2 h. Thereafter, the exposure of excess amino acids increased the phosphorylation of both S6K1 and rp S6 in a time-dependent manner. Twenty-four hours after the exposure, these phosphoryaltions were increased to the levels comparable with those seen after insulin stimulation. Protein levels of Akt, S6K1, and rp S6 were not altered during the time-course of treatment with the excess amino acids.



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  		FIG. 7. Time course of excess amino acid-induced phosphorylation of Akt, S6K1, and rp S6. Serum-starved 3T3-L1 adipocytes were incubated with standard concentrations of amino acids or with 5-fold excess of amino acids for various time periods, and the cells were stimulated with or without 17 nM insulin for 10 min. The cells were lysed and separated by SDS-PAGE and immunoblotted with anti-Ser473-phospho-specific Akt antibody (A), anti-Thr389-phosphospecific S6K1 antibody (B), and anti-phospho-specific rp S6 antibody (C). The amount of phosphorylated Akt, S6K1, and rp S6 corrected for the loaded protein amount was quantitated by densitometry. Results are the mean ± SE of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
At least two signaling pathways appear to exist including a rapamycin-sensitive pathway via the mTOR and a rapamycin-insensitive pathway in the acute regulation of protein synthesis (15, 16). Although little is known of the rapamycin-insensitive pathway, the rapamycin-sensitive pathway via mTOR is known to be activated by the nutrient as well as insulin (15, 16). Activation of mTOR leads to phosphorylation of several substrates including two critical regulators of protein synthesis, S6K1 and 4E-BP1 (15, 16). Phosphorylation of rp S6 by the activated S6K1 is associated with preferential translation of mRNAs with a polypyrimidine tract in their 5'-untranslated region (26, 27). On the other hand, the association of eIF4E with the translational complex, eIF4G, is prevented by binding of 4E-BP1 to eIF4E in the basal state. 4E-BP1 possesses four phosphorylation residues, which phosphorylation states are recognized as {alpha}-, ß-, and {gamma}-forms by Western blot analysis (29, 30). Formation of the most highly phosphorylated from, which migrates as the {gamma}-form, is associated with reduced binding to eIF4E and increased association of eIF4E with eIF4G, leading to the efficient initiation of translation (30, 31). Thus, phosphorylation of S6K1 and 4E-BP1 is associated with enhanced translation of mRNAs.

SHIP2 functions as a 5'-lipid phosphatase to hydrolyze PI3-kinase product PI(3,4,5)P3 to PI(3,4)P2 (9, 10). Overexpression of SHIP2 inhibited insulin-induced glucose uptake and glycogen synthesis via the 5'-phosphatase activity in 3T3-L1 adipocytes and L6 myocytes (10, 11). Targeted disruption of SHIP2 gene in mice increased insulin sensitivity without affecting other biological systems (12). These studies showed that SHIP2 is a physiologically important negative regulator relatively specific to insulin signaling. PDK1 is a direct target molecule of PI3-kinase, important, at least in part, for subsequent phosphorylation and activation of Akt and S6K1 (22). Consistent with previous reports, treatment with insulin did not stimulate the activity of PDK1 in 3T3-L1 adipocytes. Instead, insulin induced subcellular redistribution of PDK1 to the plasma membrane fraction, as shown in Fig. 1Go. Overexpression of SHIP2 inhibited the subcellular redistribution of PDK1. Insulin treatment also induced phosphorylation of Akt and S6K1 and rp S6, increase in the amount of 4E-BP1 {gamma}-form, and association of eIF4E with eIF4G, as shown in Figs. 2Go and 3Go. Importantly, insulin induced phosphorylation of Akt and S6K1 and rp S6, increase in the amount of 4E-BP1 {gamma}-form, and association of eIF4E with eIF4G were decreased by overexpression of SHIP2. Furthermore, overexpression of SHIP2 efficiently inhibited insulin-induced protein synthesis. These results indicate the important role of SHIP2 in the negative regulation of insulin signaling leading to the protein synthesis in addition to the glucose uptake and glycogen synthesis. We cannot rule out the possibility that other signaling elements are involved in the regulation of insulin-induced protein synthesis by SHIP2. In any case, present results further strengthen the key role of SHIP2 in the control of insulin signaling.

Oral administration of diet induces an acute rise in plasma amino acid and insulin concentrations leading to an increase in protein synthesis associated with the phosphorylation of S6K1 and 4E-BP1 (14, 15, 16, 17, 18, 19). Plasma concentrations of amino acids are known to be elevated in both rodents and human subjects with obesity, which are characterized by excess growth of adipose tissues and insulin resistance (19). Based on these facts, it would be important to clarify whether SHIP2 has a similar or distinct impact on the regulation of insulin-induced protein synthesis in the state of standard or excess concentrations of amino acids.

Interestingly, overexpression of SHIP2 inhibited insulin-induced phosphorylation of Akt, S6K1, and rp S6 similarly or more efficiently in the cell culture with 5-fold concentrations of amino acids for 30 min, compared with those seen in the standard concentrations of amino acids. Importantly, exposure of 5-fold concentrations of amino acids alone for 30 min induced transient activation of PI3-kinase activation and phosphorylation of Akt. A previous report showed that amino acids, especially leucine, promoted PI3-kinase activation in soleus muscles isolated from normal rats (13). Along this line, branched chain amino acids including leucine were the effective stimulator for the phosphorylation of Akt in 3T3-L1 adipocytes (data not shown).

The precise mechanism by which the exposure of high concentrations of amino acids for short periods only transiently induced phosphorylation of Akt is unknown. However, the effect of amino acids exposure for 30 min appears to be relatively specific to Akt in 3T3-L1 adipocytes because the amino acid exposure did not affect the phosphorylation status of serum/glucocorticoid-regulated kinase 1 (SGK1), protein kinase C {alpha}/ßII, protein kinase C-related kinase 1/2, and 90-kDa ribosomal S6 kinase as the other protein kinase A, protein kinase G, protein kinase C family (data not shown). In addition, treatment with high concentrations of amino acids for 30 min appears to elicit activation of PI3-kinase, independent of tyrosine phosphorylation of insulin receptor and IRS-1, in contrast to insulin-induced activation of PI3-kinase via insulin receptor and IRS-1 as shown in Fig. 6Go. Furthermore, tyrosine phosphorylation of IRS-1 induced by insulin stimulation for 10 min was not affected by the incubation with excess amino acids for 30 min and 16 h. Although insulin treatment for longer than 30 min induced tyrosine phosphorylation of IRS-1 appears to be reduced by the serine phosphorylation induced by the exposure of excess amino acids for more than 30 min, the serine phosphorylation does not appear to play a critical role in 10 min of insulin-induced tyrosine phosphorylation of IRS-1 (17, 33). Regardless of the precise mechanism, SHIP2 appears to effectively hydrolyze PI-kinase product generated by the treatment with high concentrations of amino acids for 30 min, leading to the efficient inhibition of phosphorylation of Akt. Alternatively, it is possible that the exposure of high concentrations of amino acids for 30 min induces adequate localization of SHIP2 for the effective hydrolysis of PI3-kinase product generated by insulin stimulation. Along this line, insulin treatment induces subcellular redistribution of SHIP2 to the plasma membrane fraction for the adequate functioning, whereas it does not alter the total 5'-phosphatase activity of SHIP2 (10, 35).

With regard to S6K1 and rp S6, they are phosphorylated to some extent even in the basal state. The exposure of the cells with 5-fold concentrations of amino acids for 30 min decreased the extent of phosphorylation of S6K1. The degree of phosphorylation of rp S6 was also decreased in subsequent time periods, as shown in Fig. 7Go. Increased insulin responsiveness in response to the decreased basal phosphorylation of S6K1 may lead to the efficient impact of SHIP2, at least in part, on the negative regulation of insulin-induced phosphorylation of S6K1 in the cell culture with the excess amino acids for 30 min. Of note is that treatment with high concentrations of amino acids for 30 min induced phosphorylation of Akt but did not lead to the phosphorylation of S6K1 and rp S6 in contrast to the insulin stimulation. The uncoupling between Akt and S6K1 in the treatment with the excess amino acids for 30 min appears to affect the impact of SHIP2 in the regulation of insulin-induced phosphorylation of S6K1 and rp S6.

In contrast to the impact of SHIP2 on the regulation of insulin-induced phosphorylation of Akt, S6K1, and rp S6 after the exposure of high concentrations of amino acids for 30 min, insulin-induced phosphorylation of S6K1 and rp S6 was not effectively affected by expression of SHIP2 in the cell culture with 5-fold concentrations of amino acids for 16 h. Interestingly, the exposure of high concentrations of amino acids alone for longer time periods elicited the phosphorylation of S6K1 and rp S6, which is not via the phosphorylation of Akt. The signaling pathway leading to protein synthesis located downstream of SHIP2 possibly via mTOR is likely to be activated by treatment with excess amino acids for 16 h. Because of the elevated of phosphorylation of S6K1 and rp S6 in the basal state after the treatment for 16 h, the effect of overexpression of SHIP2 on these phosphorylations may be less in the cells cultured with the excess amino acids for 16 h, compared with those for 30 min. However, elevation of the phosphorylation of S6K1 and rp S6 in the basal state after the treatment for 16 h alone is not completely sufficient for the cause of diminished impact of SHIP2 on the regulation of insulin-induced phosphorylation of S6K1 and rp S6. We cannot rule out the possibility that treatment with high concentrations of amino acids for 16 h affects cellular localization of SHIP2 by which the functioning of the 5'-phosphatase is not adequately elicited. In any case, it is apparent that the impact of SHIP2 on the regulation of insulin signaling leading to the protein synthesis is altered between the cells cultured with 5-fold concentrations of amino acids for 30 min and those for 16 h.

In summary, the present study indicates that SHIP2 plays an important role in the negative regulation of insulin signaling leading to the protein synthesis in 3T3-L1 adipocytes. Because the impact of SHIP2 on the insulin signaling in the cells with the acute exposure of excess concentrations of amino acids is greater than that with the chronic exposure, the impact of SHIP2 on the regulation of insulin signaling may be altered, dependent on the state with standard or excess nutrition.


   Acknowledgments
 
We thank Dr. Wataru Ogawa (Kobe University, Kobe, Japan) for kindly providing Myr-Akt and Dr. Kazuyuki Hiratani (Toyama Medical and Pharmaceutical University, Toyama, Japan) for his technical assistance.


   Footnotes
 
This work was supported in part by the Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science.

Abbreviations: DTT, Dithiothreitol; eIF, eukaryotic initiation factor; FCS, fetal calf serum; HES, buffer with HEPES, EDTA, and sucrose; IRS, insulin receptor substrate; m.o.i., multiplicity of infection; mTOR, mammalian target of rapamycin; PDGF, platelet-derived growth factor; PDK1, phosphoinositide-dependent kinase 1; PI, phosphatidylinositol; PM, plasma membrane; PMSF, phenylmethylsulfonyl fluoride; rp S6, ribosomal protein S6; SHIP2, Src homology 2-containing inositol 5'-phosphatase 2; S6K1, p70 S6 kinase; TCA, trichloroacetic acid; WT, wild- type.

Received November 20, 2003.

Accepted for publication March 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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