Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5'-phosphatase catalytic activity

Abstract

Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5'-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5'-phosphatase-defective SHIP2 (Delta IP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor beta subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or Delta IP-SHIP2. Because WT-SHIP2 possesses the 5'-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of Delta IP-SHIP2, indicating that Delta IP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase C lambda in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase C lambda, whereas these activations were increased by expression of Delta IP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of Delta IP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3beta and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of Delta IP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5'-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.

FIG. 1

Structures of SHIP2 constructs and expression in 3T3-L1 adipocytes. (A) Structures of WT-SHIP2 and 5′-phosphatase-defective SHIP2 containing Pro⁶⁸⁷-to-Ala, Asp⁶⁹¹-to-Ala, and Arg⁶⁹²-to-Gly changes are shown. The three domains of SHIP2 are an SH2 domain, a 5′-phosphatase (5′-ptase) domain, and a carboxyl-terminal proline-rich domain containing a tyrosine phosphorylation site (NPAY). (B) 3T3-L1 adipocytes were transfected with LacZ, WT-SHIP2, or ΔIP-SHIP2 at an MOI of 40 PFU/cell. Following the infection, the cells were lysed and subjected to immunoblot analysis with an anti-SHIP2 antibody. Results are representative of three separate experiments.

FIG. 2

Effect of SHIP2 overexpression on early steps of insulin signaling in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with LacZ, WT-SHIP2, or ΔIP-SHIP2 at an MOI of 40 PFU/cell. The cells were serum starved for 16 h and subsequently treated with 17 nM insulin at 37°C for the indicated times. The cell lysates were immunoprecipitated (i.p.) with anti-insulin receptor (IR) antibody (A) or anti-IRS1 antibody (B and C). The precipitates were separated by SDS–7.5% PAGE and immunoblotted with an antiphosphotyrosine antibody (A and B) or an anti-p85 subunit antibody (C). (D) The transfected 3T3-L1 adipocytes were incubated without or with insulin (17 nM) for 5 min. The cell lysates were immunoprecipitated with an antiphosphotyrosine antibody. The washed immunoprecipitates were assayed for PI 3-kinase activity with PI as the substrate, and the labeled PI(3)P product (PI3P) was resolved by thin-layer chromatography and visualized by autoradiography. Results are representative of four separate experiments.

FIG. 3

Effect of SHIP2 overexpression on generation of ³²P-labeled lipid products in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with LacZ, WT-SHIP2, or ΔIP-SHIP2 at an MOI of 40 PFU/cell. The cells were labeled with [³²P]orthophosphate (0.1 mCi/ml) for 2 h and incubated without or with insulin, and lipids were extracted with chloroform. The extracted lipids were analyzed by HPLC after being deacylated. The amounts of ³²P-labeled PI(3,4,5)P3 (A) and PI(3,4)P2 (B) generated were determined with an on-line radiochemical detector. (C) Insulin-induced generation of PI(3,4)P2 and PI(3,4,5)P3 was expressed as the ratio of PI(3,4)P2 to PI(3,4,5)P3 for each cell line within a single HPLC run. Results are representative of two separate experiments.

FIG. 4

Effect of SHIP2 overexpression on insulin-, Myr-p110-, and Myr-Akt-induced Akt activation in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes were transfected with LacZ, WT-SHIP2, or ΔIP-SHIP2 at an MOI of 40 PFU/cell. The cells were serum starved for 16 h and treated without or with insulin (17 nM) for 5 min and subsequently assayed for Akt activity. (B and C) 3T3-L1 adipocytes were transfected with either Myr-p110 (B) or Myr-Akt (C) at an MOI of 40 PFU/cell. The cells were cotransfected with LacZ, WT-SHIP2, or ΔIP-SHIP2 at the indicated MOI. The cells were serum starved for 16 h and subsequently assayed for Akt activity. The cells were lysed and immunoprecipitated with anti-Akt antibody. The washed precipitates were suspended with 200 μM ATP and 1 μg of GSK3 fusion protein and incubated for 30 min at 30°C. The samples were then separated by SDS–12% PAGE and immunoblotted with anti-Ser²¹ and -Ser⁹ phosphospecies-specific GSK3 antibody. Results are means ± SE of four separate experiments. ∗, P < 0.05 versus insulin-stimulated Akt activity in LacZ-transfected control cells (A) or versus Akt activity induced by Myr-p110 alone (B) by Student's t test.

FIG. 5

Effect of SHIP2 overexpression on insulin-, Myr-p110-, and ΔPD-induced PKCλ activation in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes were transfected with LacZ, WT-SHIP2, or ΔIP-SHIP2 at an MOI of 40 PFU/cell. The cells were serum starved for 16 h and treated without or with insulin (100 nM) for 5 min and subsequently assayed for PKCλ activity. (B and C) 3T3-L1 adipocytes were transfected with either Myr-p110 (B) or ΔPD-PKCλ (C) at an MOI of 40 PFU/cell. The cells were cotransfected with LacZ, WT-SHIP2, or ΔIP-SHIP2 at the indicated MOI. The cells were serum starvated for 16 h and subsequently assayed for PKCλ activity. The cells were lysed and immunoprecipitated with anti-PKCλ antibody. The washed immunoprecipitates were incubated for 14 min at 30°C with 0.4 μCi of [γ-³²P]ATP in a reaction mixture containing MBP as a substrate. Kinase reactions were terminated by the addition of SDS sample buffer, and samples were then fractionated by SDS-PAGE. The radioactivity incorporated into substrates was determined with a Fuji BAS 2000 image analyzer. Results are means ± SE of four separate experiments. ∗, P < 0.05 versus insulin-stimulated PKCλ activity in LacZ-transfected control cells (A) or versus PKCλ activity induced by Myr-p110 alone (B) by Student's t test.

FIG. 6

Effect of SHIP2 overexpression on insulin-induced 2-DOG uptake and Glut4 translocation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with LacZ, WT-SHIP2, or ΔIP-SHIP2 at an MOI of 40 PFU/cell. (A) Serum-starved transfected cells were stimulated with various concentrations of insulin for 15 min. Then, 2-[³H]DOG uptake for 4 min was studied. Each measurement was performed in triplicate, and results are means ± SE of six separate experiments. ∗, P < 0.05 versus 2-DOG uptake at the respective concentration of insulin in LacZ-transfected control cells by Student's t test. (B) Serum-starved transfected cells on coverslips were stimulated with various concentrations of insulin for 20 min. The cells were fixed and stained with rabbit anti-Glut4 antibody and incubated with rhodamine-conjugated anti-rabbit IgG antibody as described in Materials and Methods. The percentages of cells positive for Glut4 translocation were calculated by counting at least 300 cells at each point. Results are means ± SE of four separate experiments. ∗, P < 0.05 versus the percentages of Glut4 translocated cells at the respective concentrations of insulin in LacZ-transfected control cells by Student's t test.

FIG. 7

Effect of SHIP2 overexpression on insulin-induced GSK3β phosphorylation in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes were transfected with either LacZ, WT-SHIP2, or ΔIP-SHIP2 at an MOI of 40 PFU/cell. Serum-starved transfected cells were stimulated with 17 nM insulin for the indicated times. The cell lysates were separated by SDS–7.5% PAGE and immunoblotted with anti-Ser²¹ and -Ser⁹ phosphospecies-specific GSK3 antibody. The cell lysates were immunoblotted with an anti-GSK3 antibody (B) or an anti-SHIP2 antibody (C). (D) The amount of phosphorylated GSK3β was quantitated by densitometry. Results are means ± SE of four separate experiments. ∗, P < 0.05 versus GSK3β phosphorylation at the respective concentrations of insulin in LacZ-transfected control cells by Student's t test.

FIG. 8

Effect of SHIP2 overexpression on insulin-induced PP1 activation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with LacZ, WT-SHIP2, or ΔIP-SHIP2 at an MOI of 40 PFU/cell. The cells were serum starved for 16 h, treated without or with insulin (100 nM) for 20 min, and subsequently assayed for PP1 activity. The cells were homogenized, and PP1 activity in cell extracts toward [³²P]-labeled phosphorylase a was determined for 2 min at 37°C as described in Materials and Methods. Results are means ± SE of four separate experiments. ∗, P < 0.05 versus basal or insulin-stimulated PP1 activity in LacZ-transfected control cells by Student's t test.

FIG. 9

Effect of SHIP2 overexpression on insulin-induced glycogen synthase activation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with LacZ, WT-SHIP2, or ΔIP-SHIP2 at an MOI of 40 PFU/cell. The cells were serum and glucose starved in DMEM including 0.1% BSA and 2 mM sodium pyruvate for 3 h and then stimulated without or with insulin (17 nM) for 30 min in DMEM containing 5 mM glucose. The cells were scraped, sonicated, and centrifuged. The supernatants were resuspended in a glycogen synthase buffer containing 6.2 mM UDP-glucose, 1 mCi of [U-¹⁴C]UDP-glucose/ml, and 0.74% glycogen. The ability of the supernatant to stimulate incorporation of UDP-glucose into glycogen was determined in the absence or presence of glucose 6-phosphate (G6P; 6.2 nM). Results are expressed as mean glycogen synthase indices ± SE from four separate experiments. ∗, P < 0.05 versus insulin-stimulated glycogen synthase activity in LacZ-transfected control cells by Student's t test.

FIG. 10

Effect of SHIP2 overexpression on insulin-induced glycogen synthesis in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with LacZ, WT-SHIP2, or ΔIP-SHIP2 at an MOI of 40 PFU/cell. The cells were subsequently incubated with medium containing 5 mM glucose and 1 mCi of [¹⁴C]glucose and stimulated with various concentrations of insulin for 1 h. [¹⁴C]glucose incorporation into glycogen was analyzed as described in Materials and Methods. Results are means ± SE of six separate experiments. ∗, P < 0.05 versus glycogen synthesis at respective concentrations of insulin in LacZ-transfected control cells by Student's t test.