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Am J Physiol Endocrinol Metab 296: E157-E164, 2009. First published November 11, 2008; doi:10.1152/ajpendo.90581.2008
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Impact of lipid phosphatases SHIP2 and PTEN on the time- and Akt-isoform-specific amelioration of TNF-{alpha}-induced insulin resistance in 3T3-L1 adipocytes

Mariko Ikubo,1 Tsutomu Wada,2 Kazuhito Fukui,1 Manabu Ishiki,1 Hajime Ishihara,3 Tomoichiro Asano,4 Hiroshi Tsuneki,2 and Toshiyasu Sasaoka2

1Department of Internal Medicine and 2Department of Clinical Pharmacology, University of Toyama; 3Sainou South Hospital, Toyama; and 4Division of Molecular Medical Science, Hiroshima University, Hiroshima, Japan

Submitted 10 July 2008 ; accepted in final form 4 November 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
TNF-{alpha} is a major contributor to the pathogenesis of insulin resistance associated with obesity and inflammation by serine phosphorylating and degrading insulin receptor substrate-1. Presently, we further found that pretreatment with TNF-{alpha} inhibited insulin-induced phosphorylation of Akt2 greater than Akt1. Since lipid phosphatases SH2-containing inositol 5'-phoshatase 2 (SHIP2) and phosphatase and tensin homologs deleted on chromosome 10 (PTEN) are negative regulators of insulin's metabolic signaling at the step downstream of phosphatidylinositol 3-kinase, we investigated the Akt isoform-specific properties of these phosphatases in the negative regulation after short- and long-term insulin treatment and examined the influence of inhibition on the amelioration of insulin resistance caused by TNF-{alpha} in 3T3-L1 adipocytes. Adenovirus-mediated overexpression of WT-SHIP2 decreased the phosphorylation of Akt2 greater than Akt1 after insulin stimulation up to 15 min. Expression of a dominant-negative {Delta}IP-SHIP2 enhanced the phosphorylation of Akt2 up to 120 min. On the other hand, overexpression of WT-PTEN inhibited the phosphorylation of both Akt1 and Akt2 after short- but not long-term insulin treatment. The expression of {Delta}IP-PTEN enhanced the phosphorylation of Akt1 at 120 min and that of Akt2 at 2 min. Interestingly, the expression of {Delta}IP-SHIP2, but not {Delta}IP-PTEN, protected against the TNF-{alpha} inhibition of insulin-induced phosphorylation of Akt2, GSK3, and AS160, whereas both improved the TNF-{alpha} inhibition of insulin-induced 2-deoxyglucose uptake. The results indicate that these lipid phosphatases possess different characteristics according to the time and preference of Akt isoform-dependent signaling in the negative regulation of the metabolic actions of insulin, whereas both inhibitions are effective in the amelioration of insulin resistance caused by TNF-{alpha}.

insulin signaling; SH2-containing inositol 5'-phoshatase 2; phosphatase and tensin homologs deleted on chromosome 10

THE ACTIVATED INSULIN RECEPTOR phosphorylates insulin receptor substrates (IRS) at tyrosine residues (17, 26, 28). The tyrosine phosphorylated IRS binds to the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase), which in turn activates the p110 catalytic subunit (3, 32, 36). The activation of PI3-kinase is known to be important for the various metabolic actions of insulin (3, 33, 37). PI3-kinase functions as a lipid kinase to produce PI(3,4,5)P3 from PI(4,5)P2 in vivo (32). PI(3,4,5)P3 acts as a key lipid second messenger in insulin signaling to further downstream molecules, including Akt (30, 38). Lipid phosphatases were identified to hydrolyze PI(3,4,5)P3 in the negative regulation of insulin signaling (12, 20, 25). SH2-containing inositol 5'-phoshatase 2 (SHIP2) functions as a lipid phosphatase possessing 5'-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI(3,4)P2 (38). Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) act as a 3'-lipid phosphatase hydrolyzing PI(3,4,5)P3 to PI(4,5)P2 (20). Targeted disruption of the SHIP2 gene in mice resulted in increased insulin sensitivity and conferred protection against obesity induced by a high-fat diet (4, 33). Since homozygous disruption of the PTEN gene in mice results in embryonic lethality, the tissue-specific role of PTEN was investigated (7). Liver, skeletal muscle, or adipose tissue-specific disruption of PTEN ameliorated glucose metabolism in the animal model of diabetes (18, 34, 39); therefore, lipid phosphatases, both SHIP2 and PTEN, appear to be implicated in glucose metabolism.

Adipocytes are important target tissues of insulin, and 3T3-L1 cells are well-characterized adipocytes (22, 23, 38). Overexpression of SHIP2 and PTEN is reported to inhibit insulin-induced phosphorylation of Akt and glucose uptake (22, 23, 38); however, the effect of SHIP2 and PTEN expression has been examined only after short-term insulin treatment with controversial results (23, 35). In addition, the role of these lipid phosphatases in the regulation of metabolic signaling after long-term insulin treatment is unknown; therefore, it would be of particular importance to clarify the possible differences in characteristics and properties among these lipid phosphatases to further understand the molecular mechanism of the negative regulation of insulin signaling.

Akt is one of the downstream target molecules of PI3-kinase important for glucose metabolism (5, 6, 38). Akt1 and Akt2 are the isoforms mainly expressed in adipocytes (31, 38). Studies (5, 6) with Akt1 and Akt2 knockout mice revealed that Akt2 is preferentially implicated in glucose metabolism, whereas Akt1 is mainly involved in cell growth. The comparative effect of SHIP2 and PTEN on short- and long-term insulin-induced phosphorylation of Akt1 and Akt2 is uncertain in 3T3-L1 adipocytes. In addition, TNF-{alpha} is an important cytokine implicated in the development of insulin resistance in type 2 diabetes (14, 27, 39); therefore, investigation of the ameliorative effect by inhibition of these endogenous lipid phosphatases on TNF-{alpha}-induced insulin resistance is important to clarify the therapeutic value in type 2 diabetes.

In the present study, we directly compared the role of lipid phosphatases SHIP2 and PTEN in short- and long-term insulin-induced phosphorylation of Akt1 and Akt2 in 3T3-L1 adipocytes. Furthermore, we investigated whether the inhibition of endogenous SHIP2 and PTEN by phosphatase-defective mutant expression protects against the impairment of insulin-induced phosphorylation of Akt, GSK3, and Akt substrate 160 (AS160), and glucose uptake by pretreatment with TNF-{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Human crystal insulin was provided by Novo Nordisk Pharmaceutical (Copenhagen, Denmark). 2-[3H]deoxyglucose (2DG; 3,330 GBq/mM) was purchased from NEN Life Science Products (Boston, MA). Human recombinant TNF-{alpha} was obtained from Pepro Tech (Rocky Hill, NJ). The two polyclonal anti-SHIP2 antibodies were described previously (12). A monoclonal anti-phosphotyrosine antibody (PY20) was purchased from Transduction Laboratories (Lexington, KY). A polyclonal anti-Thr308/309 phospho-specific Akt antibody and a polyclonal anti-Ser473/474 phospho-specific Akt antibody, a polyclonal anti-Akt antibody, a polyclonal anti-Akt2 antibody, a polyclonal anti-Ser21/9 phospho-specific GSK3{alpha}/β antibody, a polyclonal anti-GSK3{alpha}/β antibody, an a polyclonal anti-Ser/Thr-phospho-specific Akt substrate antibody, and a polyclonal anti-AS160 antibody were from Cell Signaling (Beverly, MA). A monoclonal anti-Akt1 antibody and a monoclonal anti-PTEN antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). A polyclonal anti-IRS-1 antibody was from Upstate Biotechnology (Lake Placid, NY). Enhanced chemiluminescence reagents were from GE Healthcare Bio-Science (Tokyo, Japan). DMEM was from GIBCO-BRL Japan (Tokyo, Japan). All other reagents were of analytical grade and purchased from Sigma Chemical (St. Louis, MO) or Wako Pure Chemical Industries (Osaka, Japan).

Adenoviral vectors. Adenoviral vectors encoding wild-type SHIP2 (WT-SHIP2), a phosphatase-defective mutant SHIP2 ({Delta}IP-SHIP2) containing Pro687 to Ala, Asp691 to Ala, and Arg692 to Gly changes (38), wild-type PTEN (WT-PTEN), and a phosphatase-defective mutant PTEN ({Delta}IP-PTEN) containing Cys124 to Ser change (23) were described previously.

Cell culture and infections with adenovirus. 3T3-L1 fibroblasts were grown and passaged in DMEM supplemented with 10% donor calf serum. Cells at 2–3 days postconfluence were used for differentiation. The differentiation medium contained 10% FBS, 250 nM dexamethazone, 0.5 mM IBMX, and 500 nM insulin. After 3 days, the differentiation medium was replaced with postdifferentiation medium containing 10% FBS and 500 nM insulin. After 3 more days, the postdifferentiation medium was replaced with DMEM including 25 mM glucose supplemented with 10% FBS. SHIP2 and PTEN were transiently expressed in differentiated 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. A multiplicity of infection (MOI) of 10–40 plaque-forming units (PFU)/cell was used to infect 3T3-L1 adipocytes in DMEM containing 2% FBS, 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 (38). The efficiency of adenovirus-mediated gene transfer of SHIP2 and PTEN was ~95%.

Insulin and TNF-{alpha} treatment. 3T3-L1 adipocytes grown in six-well multiplates were incubated with DMEM containing 0.1% FBS without or with 17 nM insulin at 37°C for 2- to 120-min periods. For experiments with TNF-{alpha} treatment, 20 nM TNF-{alpha} were added for 16 h and then treated with 17 nM insulin for 10 or 120 min.

Immunoprecipitation and Western blotting. The cells were lysed in a buffer containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium deoxycholate, 1 mM β-glycerophosphate, 1% NP40, 1 mM PMSF, 1 mM Na3VO4, 50 mM sodium fluoride, 10 µg/ml of aprotinin, and 10 µM leupeptin, pH 7.4, for 30 min at 4°C. The lysates were centrifuged to remove insoluble materials. The supernatants (100 µg of protein) were immunoprecipitated with antibodies for 2 h at 4°C. The precipitates or the lysates were then separated by 7.5% SDS-PAGE and transferred onto polyvinylidene difluoride membranes using a Bio-Rad Transblot apparatus. 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. They were then probed with antibodies for 2 h at 20°C or for 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, the blots were incubated with a horseradish peroxidase-linked secondary antibody and subjected to enhanced chemiluminescence detection using ECL reagent according to the manufacturer's instructions (GE Health Science Bio-Science; Ref. 38). Densitometric analysis was conducted directly from the blotted membrane by utilizing LAS-4000 lumino-image analyzer system (Fujifilm, Tokyo, Japan). The relative phosphorylation level of each protein was calculated as the ratio of phosphorylated to total protein level.

Measurement of 2-deoxyglucose uptake. 3T3-L1 adipocytes grown in six-well multiplates were pretreated with TNF-{alpha} and serum starved for 2 h. The cells were washed twice with PBS and incubated with Krebs-Ringer phosphate-HEPES buffer, 10 nM HEPES, 131.2 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, and 2.5 mM NaH2PO4, containing 1% BSA, pH 7.4, for 1 h at 37°C. The cells were subsequently stimulated with various concentrations of insulin. After a 15-min insulin treatment, 3.7 kBq of 2-[3H]deoxyglucose (2-DG) were added for 4 min. The reaction was stopped by the addition of 10 µnol/l cytochalasin B. The cells were washed three times with PBS and solubilized with 0.2 mM SDS-0.2 N NaOH (38). The radioactivity incorporated into the cells was measured by liquid scintillation counting.

Statistical analysis. Data are expressed as means ± SE. P values were determined by one-way ANOVA with Bonferroni's correction test, and P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of lipid phosphatases in 3T3-L1 adipocytes. SHIP2 (140-kDa) is a 5'-lipid phosphatase and PTEN (54-kDa) is a 3'-lipid phosphatase, both of which are known to be involved in the negative regulation of insulin signaling (22, 23, 30, 38). Endogenous SHIP2 and PTEN were clearly observed in control 3T3-L1 adipocytes. Consensus amino acids located within the catalytic domain of lipid phosphatases were mutated to generate phosphatase-defective SHIP2 ({Delta}IP-SHIP2) and PTEN ({Delta}IP-PTEN) (23, 38). Wild-type and phosphatase-defective lipid phosphatases were transiently expressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. By transfecting with these lipid phosphatase genes at an MOI of 10 PFU/cell (Fig. 1, A and B) and 40 PFU/cell (Fig. 1, C and D), we observed a 2.5- and 5-fold increase in expression levels of SHIP2 and a 4- and 7-fold increase in expression levels of PTEN, respectively, over the endogenous level in 3T3-L1 adipocytes. Since the obtained results with expression at an MOI of 10 and 40 PFU/cell were similar, the after analyses were shown at an MOI of 40 PFU/cell.


Figure 1
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  		Fig. 1. Expression of lipid phosphatases in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with LacZ, wild-type SH2-containing inositol 5'-phosphatase 2 (WT-SHIP2), phosphatase-defective mutant SHIP2 ({Delta}IP-SHIP2), wild-type phosphatase and tensin homologs deleted on chromosome 10 (WT-PTEN), and {Delta}IP-PTEN at a multiplicity of infection (MOI) of 10 plaque-forming units (PFU)/cells (A and B) and 40 PFU/cells (C and D). After infection, cells were lysed and subjected to immunoblot analysis with anti-SHIP2 antibody (A and C) and anti-PTEN antibody (B and D). Results represent 3 separate experiments.

 
Effect of expression of wild-type lipid phosphatases on insulin-induced phosphorylation of Akt. Akt is one of the downstream target molecules of PI3-kinase and has been shown to mediate the metabolic actions of insulin (5, 38). We compared the effect of WT-SHIP2 and WT-PTEN expressions on the phosphorylation of Akt1 and Akt2 after insulin stimulation for up to 120 min in 3T3-L1 adipocytes. Overexpression of WT-SHIP2 mildly inhibited the phosphorylation of Akt1 at Thr308 after insulin stimulation for up to 120 min (13.0% reduction at 5 min; Fig. 2A). On the other hand, overexpression of WT-SHIP2 greatly inhibited Akt2 phosphorylation at Thr309 after insulin treatment for up to 15 min (30.9% reduction at 5 min; Fig. 2C). Overexpression of WT-PTEN significantly inhibited insulin-induced Akt1 phosphorylation at 2 and 5 min by 26.6 and 16.5%, respectively (Fig. 2B). Insulin-induced phosphorylation of Akt2 at 2 and 5 min was also inhibited by 29.1 and 20.0%, respectively, by the overexpression of WT-PTEN (Fig. 2D). Similar results were obtained concerning Akt phosphorylation at Ser473/474 residue and GSK3 phosphorylation at Ser21/9 residue (data not shown).


Figure 2
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  		Fig. 2. Effect of wild-type SHIP2 and PTEN overexpression on insulin-induced phosphorylation of Akt. 3T3-L1 adipocytes were transfected with WT-SHIP2 (A and C) and WT-PTEN (B and D) at an MOI of 40 PFU/cell. Cells were serum starved for 16 h and subsequently treated with 17 nM insulin at 37°C for indicated times. Cells were immunoprecipitated with anti-Akt1 antibody (A and B) or anti-Akt2 antibody (C and D). Precipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-Thr308/309-phospho-specific Akt antibody, anti-Akt1 antibody, or anti-Akt2 antibody. Amount of Akt phosphorylated at Thr308/309 corrected for total protein level was quantitated by densitometry. Results are means ± SE of 5 separate experiments. *P < 0.05 vs. amount of phosphorylated Akt in LacZ-transfected cells with respective insulin treatment.

 
Effect of expression of phosphatase-defective ({Delta}IP) lipid phosphatases on insulin-induced phosphorylation of Akt. Expression of {Delta}IP-SHIP2 enhanced Akt1 phosphorylation at Thr308, and phosphorylation was significantly enhanced by 70.4% after 120 min of insulin treatment (Fig. 3A). The enhancing effect was more apparent in Akt2 phosphorylation at Thr309. Phosphorylation of Akt2 at 2, 5, and 120 min of insulin treatment was significantly augmented by 20.7, 16.6, and 93.7%, respectively, by the expression of {Delta}IP-SHIP2 (Fig. 3C). Expression of {Delta}IP-PTEN enhanced long-term insulin-stimulated Akt1 phosphorylation, which was significantly augmented by 43.9% after 120 min of insulin treatment (Fig. 3B). Phosphorylation of Akt2 at only 2 min of insulin stimulation was slightly enhanced by 17.7% by the expression of {Delta}IP-PTEN (Fig. 3D). Similar results were again obtained concerning the phosphorylation of Akt at Ser473/474 residue and GSK3 at Ser21/9 residue (data not shown).


Figure 3
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  		Fig. 3. Effect of phosphatase-defective SHIP2 and PTEN expression on insulin-induced phosphorylation of Akt. 3T3-L1 adipocytes were transfected with {Delta}IP-SHIP2 (A and C) and {Delta}IP-PTEN (B and D) at an MOI of 40 PFU/cell. Cells were serum starved for 16 h and subsequently treated with 17 nM insulin at 37°C for indicated times. Cells were immunoprecipitated with anti-Akt1 antibody (A and B) or anti-Akt2 antibody (C and D). Precipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-Thr308/309-phospho-specific Akt antibody. Amount of Akt phosphorylated at Thr308/309 corrected for total protein level was quantitated by densitometry. Results are means ± SE of 5 separate experiments. *P < 0.05 vs. amount of phosphorylated Akt in LacZ-transfected cells with respective insulin (Ins.) treatment.

 
Expression of phosphatase-defective lipid phosphatases ameliorates insulin-induced phosphorylation of Akt after TNF-{alpha} treatment. We next examined the ameliorative effect of the expression of phosphatase-defective lipid phosphatases on the decreased insulin-induced phosphorylation of Akt by pretreatment with TNF-{alpha} (Fig. 4). Pretreatment with TNF-{alpha} decreased the phosphorylation of Akt1 at Thr308 after insulin stimulation for 10 min (lane 5 vs. 7) but not for 120 min (lane 9 vs. 11). The TNF-{alpha}-induced decrease in insulin-induced phosphorylation of Akt1 at 10 min was not apparently affected by the expression of either {Delta}IP-SHIP2 (Fig. 4A) or {Delta}IP-PTEN (Fig. 4B). In contrast, the expression of either phosphatase-defective mutant enhanced insulin-induced phosphorylation of Akt1 at 120 min even after pretreatment with TNF-{alpha} (Fig. 3, A and B, and Fig. 4, A and B). On the other hand, pretreatment with TNF-{alpha} markedly inhibited the insulin-induced phosphorylation of Akt2 (Thr309 residue) at 10 min (lane 5 vs. 7) and also at 120 min (lane 9 vs. 11). The decreased phosphorylation of Akt2 by pretreatment with TNF-{alpha} was effectively ameliorated by the expression of {Delta}IP-SHIP2 (Fig. 4C) but not by the expression of {Delta}IP-PTEN (Fig. 4D). Similar results were obtained by the reciprocal immunoblotting studies with anti-Akt1 antibody or anti-Akt2 antibody after immunoprecipitation with anti-Thr308/309 phospho-specific Akt antibody (data not shown). In addition, similar findings were again observed concerning the phosphorylation of Akt at Ser473/474 residue (data not shown).


Figure 4
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  		Fig. 4. Effect of phosphatase-defective SHIP2 and PTEN expression on TNF-{alpha}-induced decrease in phosphorylation of Akt. 3T3-L1 adipocytes were transfected with {Delta}IP-SHIP2 (A and C) and {Delta}IP-PTEN (B and D) at an MOI of 40 PFU/cell. Serum-starved transfected cells preincubated with 20 nM TNF-{alpha} for 16 h were treated with 17 nM insulin for 10 and 120 min. Cells were immunoprecipitated with anti-Akt1 antibody (A and B) or anti-Akt2 antibody (C and D). Precipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-Thr308/309-phospho-specific Akt antibody. Amount of phosphorylated Akt at Thr308/309 corrected for total protein level was quantitated by densitometry. Results are means ± SE of 4 separate experiments. *P < 0.05 vs. amount of phosphorylated Akt in LacZ-transfected cells with respective insulin treatment.

 
Expression of phosphatase-defective lipid phosphatases ameliorates insulin-induced phosphorylation of GSK3 and AS160 after TNF-{alpha} treatment. GSK3 and AS160 are important downstream molecules of Akt implicated in the metabolic action of insulin (3, 28, 37). We therefore investigated the ameliorative effect of the expression of phosphatase-defective lipid phosphatases on the decreased insulin-induced phosphorylation of GSK3 and AS160 by pretreatment with TNF-{alpha} (Fig. 5). Pretreatment with TNF-{alpha} decreased the phosphorylation of GSK3 and AS160 after insulin stimulation for 10 min (lane 5 vs. 7) and for 120 min (lane 9 vs. 11). TNF-{alpha}-induced decrease in insulin-induced phosphorylation of both GSK3 and AS160 was relatively more ameliorated by the expression of {Delta}IP-SHIP2 (Fig. 5, A and C) than that of {Delta}IP-PTEN (Fig. 5, B and D). These results indicate that the change of GSK3 and AS160 phosphorylations is relatively correlated with the phosphorylation of Akt2 rather than Akt1 by the expression of {Delta}IP-SHIP2.


Figure 5
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  		Fig. 5. Effect of phosphatase-defective SHIP2 and PTEN expression on TNF-{alpha}-induced decrease in phosphorylation of GSK3 and AS160. 3T3-L1 adipocytes were transfected with {Delta}IP-SHIP2 (A and C) and {Delta}IP-PTEN (B and D) at an MOI of 40 PFU/cell. Serum-starved transfected cells preincubated with 20 nM TNF-{alpha} for 16 h were treated with 17 nM insulin for 10 and 120 min. Total cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-Ser21/9-phospho-specific GSK3{alpha}/β antibody (A and B), anti-GSK3{alpha}/β antibody (A and B), anti-Ser/Thr-phospho-specific Akt substrate antibody (C and D), or anti-AS160 antibody (C and D). Amount of phosphorylated GSK3 and AS160 corrected for total GSK3 and AS160 levels, respectively, was quantitated by densitometry. Results are expressed as means ± SE of 4 separate experiments. *P < 0.05 vs. amount of phosphorylated GSK3 or AS160 in LacZ-transfected cells with respective insulin treatment.

 
Expression of phosphatase-defective lipid phosphatases does not affect insulin-induced degradation and tyrosine phosphorylation of IRS-1 after TNF-{alpha} treatment. We further examined whether expression of phosphatase-defective lipid phosphatases affects insulin-induced degradation and tyrosine phosphorylation of IRS-1 after TNF-{alpha} treatment, since TNF-{alpha} is an important adipokine that causes insulin resistance by facilitating the degradation of IRS-1 in 3T3-L1 adipocytes (14). As a result, insulin-induced tyrosine phosphorylation of IRS-1 is decreased after pretreatment with TNF-{alpha} (14). Consistent with these findings, pretreatment with TNF-{alpha} caused the degradation of IRS-1, and the subsequent tyrosine phosphorylation of IRS-1 after insulin treatment for 10 and 120 min was decreased. The expression of neither {Delta}IP-SHIP2 (Fig. 6A) nor {Delta}IP-PTEN (Fig. 6B) affected TNF-{alpha}- and insulin-induced degradation of IRS-1. Similarly, the suppressing effect of TNF-{alpha} on insulin-induced tyrosine phosphorylation of IRS-1 at 10 and 120 min was not affected by the expression of either {Delta}IP-SHIP2 (Fig. 6C) or {Delta}IP-PTEN (Fig. 6D). These results indicate that the expression of {Delta}IP-SHIP2 and {Delta}IP-PTEN ameliorates the decreased Akt-dependent signaling without affecting degradation of IRS-1. It is of note that treatment with TNF-{alpha} itself also did not affect the expression of endogenous SHIP2 and PTEN (data not shown).


Figure 6
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  		Fig. 6. Effect of phosphatase-defective SHIP2 and PTEN expression on insulin-induced degradation and tyrosine phosphorylation of IRS-1 after pretreatment with TNF-{alpha}. 3T3-L1 adipocytes were transfected with {Delta}IP-SHIP2 (A and C) and {Delta}IP-PTEN (B and D) at an MOI of 40 PFU/cell. Cells were serum starved for 16 h and subsequently pretreated with 20 ng/ml of TNF-{alpha} for 16 h. They were then treated with 17 nM insulin at 37°C for 10 and 120 min. Cells were lysed and separated by 7.5% SDS-PAGE and immunoblotted with anti-IRS-1 antibody (A and B) or anti-phosphotyrosine antibody (C and D). Amount of IRS protein and tyrosine-phosphorylated IRS-1 was quantitated by densitometry. Results are means ± SE of 4 separate experiments. *P < 0.05 vs. amount of IRS in LacZ-transfected cells with respective insulin treatment.

 
Effect of expression of phosphatase-defective lipid phosphatases on insulin-induced glucose uptake after TNF-{alpha} treatment. We finally examined the effect of the expression of phosphatase-defective lipid phosphatases on insulin-induced 2-DG uptake in the presence or absence of TNF-{alpha} (Fig. 7). The expression of either {Delta}IP-SHIP2 or {Delta}IP-PTEN enhanced insulin-induced 2-DG uptake, although the former augmented it greater than the latter. Treatment with TNF-{alpha} inhibited 1.7- and 17-nM insulin-induced 2-DG uptake by 42.7 and 19.0%, respectively. Interestingly, the expression of either {Delta}IP-SHIP2 or {Delta}IP-PTEN ameliorated the reduced 2-DG uptake by pretreatment with TNF-{alpha} to the control level. The amount of GLUT4 protein was not altered by treatment with TNF-{alpha} or the expression of both {Delta}IP-SHIP2 and {Delta}IP-PTEN (data not shown).


Figure 7
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  		Fig. 7. Effect of phosphatase-defective SHIP2 and PTEN expression on TNF-{alpha}-induced inhibition of glucose uptake. 3T3-L1 adipocytes were transfected with {Delta}IP-SHIP2 or {Delta}IP-PTEN at an MOI of 40 PFU/cell. Serum-starved transfected cells pretreated with 20 nM TNF-{alpha} for 16 h were incubated in glucose-free medium for 30 min. After the cells had been stimulated with 10 nM insulin for 15 min, 3.7 kBq of 2-[3H]deoxyglucose (2-[3H]DG) were added for 4 min. Reaction was stopped by the addition of 10 µM cytochalasin B. Cells were washed 3 times with PBS and solubilized with 0.2 mM SDS-0.2 N NaOH. Radioactivity incorporated into the cells was measured with a liquid scintillation counter. Results are means ± SE of 5 separate experiments. *P < 0.05 vs. 2-[3H]DG uptake in LacZ-transfected control cells with respective insulin treatment. {Phi}P < 0.05 vs. 2-[3H]DG uptake in TNF-{alpha}-treated cells with respective insulin treatment.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
SHIP2 and PTEN are lipid phosphatases known to be involved in the negative regulation of insulin signaling in vivo and/or in vitro (4, 18, 20, 23, 33–35, 38, 39); however, the possible difference in characteristics and properties among these lipid phosphatases after short- vs. long-term insulin treatment is still unknown. Our previous reports (31) showed that SHIP2 predominantly regulates the phosphorylation of Akt2, but not Akt1, after short-term insulin treatment. Again, our current results showed that overexpression of WT-SHIP2 inhibited insulin-induced phosphorylation of Akt2 rather than Akt1 only after insulin treatment for up to 15 min. Interestingly, the effect was diminished after 60 and 120 min of insulin stimulation. Furthermore, the expression of {Delta}IP-SHIP2 enhanced insulin-induced phosphorylation of Akt2 after both short- and long-term insulin treatments, whereas phosphorylation of Akt1 was augmented only after 120 min of insulin treatment by the expression; therefore, the present results further clarified the characteristics of the regulation of insulin signaling by SHIP2, indicating that SHIP2 predominantly regulates the phosphorylation of Akt2 rather than Akt1 in a time-specific manner.

PTEN is a 3'-lipid phosphatase hydrolyzing PI(3,4,5)P3 to PI(4,5)P2 (20). It is reported that overexpression of WT-PTEN inhibited short-term insulin-induced activation of Akt, although the isoform-specific difference was not examined (22, 23, 35). On the other hand, the expression of {Delta}IP-PTEN did not affect short-term insulin-induced metabolic signaling, whereas the amount of PI(3,4,5)P3 was increased in 3T3-L1 adipocytes (23). In contrast, depletion of PTEN protein by siRNA-mediated gene silencing enhanced short-term insulin-induced phosphorylation of Akt (35). The present studies showed that overexpression of WT-PTEN inhibited the phosphorylation of Akt1 and Akt2 after insulin stimulation for up to 5 min and that the effect was diminished thereafter. Our results clearly indicate that the effect of WT-PTEN expression is not specific to either Akt1 or Akt2 and is seen only after short-term insulin treatment. In addition, the expression of {Delta}IP-PTEN enhanced Akt2 phosphorylation, and only had a mild impact on augmenting the phosphorylation of Akt1 after 2 min of insulin treatment. Furthermore, {Delta}IP-PTEN expression enhanced the phosphorylation of Akt1, but not Akt2, after 120 min of insulin treatment. Taken together, PTEN appears to be implicated in the regulation of both short- and long-term insulin treatment-induced phosphorylation of Akt, whereas the Akt isoform-specific difference during regulation is ambiguous. SHIP2 is reported to translocate from cytosol to plasma membrane whereby phosphorylation of Akt2 is preferentially regulated upon insulin treatment (31), whereas the redistribution of PTEN is uncertain in 3T3-L1 adipocytes. The possible difference of subcellular redistribution between SHIP2 and PTEN may be a reason to cause an alteration in the isoform and temporal specific effects of Akt phosphorylation. Alternatively, it is possible that the stronger effect of SHIP2 on Akt2 and the equivalent effects of PTEN on Akt1 and Akt2 reflect different properties of the pleckstrin homology (PH) domain of isoforms. Along this line, the PH domain of Akt2 may have higher affinity to PI(3,4,5)P3 than PI(3,4)P2, whereas the PH domain of Akt1 has similar affinities to both PI(3,4,5)P3 and PI(3,4)P2.

SHIP2 appears to be implicated in insulin resistance as a cause of type 2 diabetes in addition to the control of glucose homeostasis (4, 15, 16, 21, 33). SHIP2 knockout mice demonstrated enhanced phosphorylation of Akt in the skeletal muscle and liver, whereas whole body glucose homeostasis is not altered in mice fed a normal chow diet (33). However, the mice were protected from obesity and insulin resistance caused by a high-fat diet (33). Consistent with the report, the liver-specific inhibition of endogenous SHIP2 via the adenovirus-mediated expression of {Delta}IP-SHIP2 ameliorated glucose metabolism and insulin resistance in diabetic db/db mice and KK-Ay mice (8, 9). In addition, muscle denervation is known to cause insulin resistance characterized by a decrease in the ability of insulin to stimulate glucose uptake and glycogen synthesis in rats (1). A reduction of SHIP2 expression using an antisense oligonucleotide against SHIP2 mRNA ameliorated insulin resistance in rats (1). Furthermore, insulin resistance caused by chronic insulin treatment was effectively ameliorated by the expression of {Delta}IP-SHIP2 (29). Taken together, inhibition of endogenous SHIP2 appears to be valuable in the amelioration of insulin resistance in type 2 diabetes. Concerning the pathological impact of PTEN in glucose homeostasis (10, 13), heterozygous deletion of the PTEN gene in IRS-2 knockout mice conferred protection from insulin resistance, although homozygous disruption of the PTEN gene in mice resulted in embryonic lethality (19). Antisense oligonucleotide-mediated inhibition of endogenous PTEN expression in the liver led to the amelioration of elevated glucose levels and decreased insulin sensitivity in diabetic ob/ob and db/db mice (2). Adipose tissue-specific knockout of PTEN is known to protect against streptozotocin-induced diabetes (18). Muscle-specific knockout of PTEN resulted in the amelioration of decreased insulin-induced phosphorylation of Akt in the soleus caused by high-fat feeding (39). Although tissue-specific inhibition of PTEN may also appear to be a therapeutic target in the treatment of type 2 diabetes with insulin resistance, the main role of PTEN is the regulation of cell growth and tumor suppressor (7, 18, 34, 39); therefore, care should be taken when inhibiting PTEN for therapeutic usage because of possible tumor formation. Taken together, it is important to clarify the different molecular mechanisms by which inhibition of these lipid phosphatases ameliorates the state of insulin resistance.

TNF-{alpha} is an important adipokine causing insulin resistance by impairing insulin signaling (14, 27, 40). In the present study, we clarified that treatment with TNF-{alpha} impaired insulin-induced phosphorylation of Akt2 more profoundly than Akt1 in 3T3-L1 adipocytes. Interestingly, the expression of dominant-negative SHIP2 and PTEN enhanced the phosphorylation of Akt1 induced by insulin treatment for 120 min together with TNF-{alpha}. The present results indicate that enhancement of PI3-kinase-dependent insulin signaling by the inhibition of either lipid phosphatase is sufficient to ameliorate insulin-induced phosphorylation of Akt. Interestingly, inhibition of SHIP2, but not PTEN, effectively restored the impaired phosphorylation of Akt2 caused by TNF-{alpha} treatment. Similarly, the impaired phosphorylation of GSK3 and AS160 was ameliorated relatively more effectively by inhibition of SHIP2 than that of PTEN. Since Akt2 rather than Akt1 is closely related to the control of glucose metabolism (5), inhibition of SHIP2 rather than PTEN might be a more suitable approach to ameliorate decreased metabolic signaling of insulin in the state of insulin resistance; however, inhibition of either SHIP2 or PTEN ameliorated reduced insulin-induced glucose uptake caused by TNF-{alpha} to the same extent. It is uncertain why the extent of amelioration in glucose uptake is similar, whereas inhibition of SHIP2 improved the phosphorylation of Akt2 more than PTEN. Amelioration of Akt1 phosphorylation may be sufficient to improve glucose uptake, at least under our experimental conditions. Alternatively, the PI(3,4,5)P3-mediated pathway independent of Akt may regulate glucose uptake. It might be necessary to examine the effect of glucose uptake more precisely to dissect the ameliorative role of inhibition between SHIP2 and PTEN.

In summary, the present results indicate that SHIP2 predominantly regulates insulin-induced phosphorylation of Akt2 rather than Akt1 in a time-specific manner and that PTEN regulates insulin-induced phosphorylation of both Akt1 and Akt2, whereas isoform specificity is ambiguous in the regulation in 3T3-L1 adipocytes. In addition, the expression of the dominant-negative SHIP2 effectively ameliorated decreased phosphorylation of Akt2 caused by pretreatment with TNF-{alpha}, whereas the reduced phosphorylation of Akt1 was restored by the expression of either of these phosphatase-defective lipid phosphatases without affecting the degradation of IRS-1. Our results further extend the knowledge that inhibition of both SHIP2 and PTEN is an attractive approach to ameliorate the metabolic action of insulin in the state of insulin resistance, whereas inhibition of SHIP2 appears to have more impact than PTEN on the amelioration of Akt2 phosphorylation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to T. Sasaoka).


   ACKNOWLEDGMENTS
 
We thank Dr. Kazuyuki Tobe and Dr. Masashi Kobayashi for support (University of Toyama, Toyama, Japan).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Sasaoka, Dept. of Clinical Pharmacology, Univ. of Toyama, 2630 Sugitani, Toyama 930-0194, Japan (e-mail: tsasaoka{at}pha.u-toyama.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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