Inhibition of endogenous SHIP2 ameliorates insulin resistance caused by chronic insulin treatment in 3T3-L1 adipocytes
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SHIP2 is a physiologically important negative regulator of insulin signalling hydrolysing the PI3-kinase product, PI(3,4,5)P3, which also has an impact on insulin resistance. In the present study, we examined the effect of inhibiting the endogenous SHIP2 function on the insulin resistance caused by chronic insulin treatment.
The endogenous function of SHIP2 was inhibited by expressing a catalytically inactive SHIP2 (ΔIP-SHIP), and compared with the effect of treatments designed to restore the levels of IRS-1 in insulin signalling systems of 3T3-L1 adipocytes.
Chronic insulin treatment induced the large (86%) down-regulation of IRS-1 and the modest (36%) up-regulation of SHIP2. Subsequent stimulation by insulin of Akt phosphorylation, PKCλ activity, and 2-deoxyglucose (2-DOG) uptake was markedly decreased by the chronic insulin treatment. Coincubation with the mTOR inhibitor, rapamycin, effectively inhibited the proteosomal degradation of IRS-1 caused by the chronic insulin treatment. Although the coincubation with rapamycin and advanced overexpression of IRS-1 effectively ameliorated subsequent insulin-induced phosphorylation of Akt, insulin stimulation of PKCλ activity and 2-DOG uptake was partly restored by these treatments. Similarly, expression of ΔIP-SHIP2 effectively ameliorated the insulin-induced phosphorylation of Akt without affecting the amount of IRS-1. Furthermore, the decreased insulin-induced PKCλ activity and 2-DOG uptake following chronic insulin treatment were ameliorated by the expression of ΔIP-SHIP2 more effectively than by the treatment with rapamycin.
Our results indicate that the inhibition of endogenous SHIP2 is effective in improving the state of insulin resistance caused by chronic insulin treatment.
KeywordsAkt Glucose uptake Insulin Insulin resistance PKCλ SHIP2
glucose transporter 4
insulin receptor substrate-1
mammalian target of rapamycin
platelet-derived growth factor
protein kinase C
SH2-containing inositol 5′-phosphatase 2
The activation of phosphatidylinositol 3-kinase (PI3-kinase) is known to be important to the various metabolic actions of insulin [1, 2, 3, 4]. PI(3,4,5)P3 produced by activated PI3-kinase is thought to function as a key lipid second messenger in insulin signalling to further downstream molecules [3, 4, 5]. We and others identified SH2-containing inositol 5′-phosphatase 2 (SHIP2) as a lipid phosphatase possessing 5′-phosphatase activity to hydrolyse PI(3,4,5)P3 to PI(3,4)P2 [6, 7]. Previous reports have indicated that overexpression of SHIP2 inhibits insulin-induced glucose uptake and glycogen synthesis via its 5′-phosphatase activity in 3T3-L1 adipocytes and L6 myocytes [8, 9]. Targeted disruption of the SHIP2 gene in mice increased sensitivity to insulin without affecting other biological systems . These findings indicate that SHIP2 is a physiologically important negative regulator that is relatively specific to insulin signalling. In addition, expression of SHIP2 protein is enhanced in the skeletal muscle and fat tissue of diabetic db/db mice . Treatment with the insulin-sensitizing thiazolidinedione, rosiglitazone, lowered the elevated levels of SHIP2 in the db/db mice . Furthermore, a deletion in the 3′ untranslated region within the motifs implicated in the control of protein synthesis leading to the possible increase in expression of SHIP2 protein was identified in the UK and Belgian population of individuals with type 2 diabetes . Therefore, SHIP2 is implicated in insulin resistance as a cause of type 2 diabetes in addition to the physiological importance in insulin signalling. Based on these findings, inhibition of endogenous SHIP2 function may be a target for ameliorating insulin signalling in the state of insulin resistance.
Hyperinsulinaemia is a hallmark of insulin resistance [13, 14, 15]. Chronic hyperinsulinaemia causes a desensitization to subsequent insulin responses, which appears to be part of the vicious cycle involved in the pathogenesis of type 2 diabetes [16, 17, 18]. In this regard, chronic treatment with insulin is known to facilitate the proteosomal degradation of IRS-1 leading to the down-regulation of insulin signalling at IRS-1 in 3T3-L1 adipocytes [17, 18, 19]. However, it is unknown whether SHIP2 is also involved in the resistance caused by chronic exposure to insulin. In the present study, the change in SHIP2 expression following chronic insulin treatment was investigated in 3T3-L1 adipocytes. In addition, the effect of inhibition of endogenous SHIP2 function using adenovirus-mediated gene transfer of a dominant-negative SHIP2 (ΔIP-SHIP2) on the possible amelioration of decreased insulin signalling caused by the chronic insulin treatment was investigated. The down-regulation of insulin signalling at the level of IRS-1 caused by the chronic insulin treatment can be ameliorated by pretreatment with rapamycin, which is an inhibitor of mTOR-dependent proteosomal degradation of IRS-1 [20, 21]. Alternatively, the decrease of IRS-1 can be prevented by overexpression of IRS-1 through adenovirus-mediated gene transfer . Finally, the effects of the amelioration at the level of IRS-1 and SHIP2 on the chronic insulin treatment-induced down-regulation of insulin signalling were compared.
Materials and methods
Human crystal insulin was provided by Novo Nordisk Pharmaceutical (Copenhagen, Denmark). [γ-32P]ATP (111 TBq/mmol) and 2-[3H]deoxyglucose (DOG; 3,330 GBq/mmol) were purchased from NEN Life Science Products (Boston, MA, USA). The two polyclonal anti-SHIP2 antibodies were described previously . A polyclonal anti-PKCλ antibody was kindly provided by Dr W. Ogawa (Kobe University, Japan) . A monoclonal antiphosphotyrosine antibody (PY99) was from Transduction Laboratories (Lexington, KY, USA). A polyclonal anti-Thr308 phospho-specific Akt antibody, a polyclonal anti-Ser473 phospho-specific Akt antibody, and a monoclonal anti-PKCλ antibody were from Cell Signalling (Beverly, MA, USA). A polyclonal anti-Akt antibody and a polyclonal anti-Glut4 antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A polyclonal IRS-1 antibody and a polyclonal anti-PDGF β receptor antibody were from Upstate Biotechnology (Lake Placid, NY, USA). Enhanced chemiluminescence reagents were from Amersham Pharmacia Biotech (Uppsala, Sweden). Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium (MEM) vitamin mixtures, and MEM amino acid solutions were from Gibco BRL Japan (Tokyo, Japan). All other reagents were of analytical grade and purchased from Sigma Chemical (St Louis, MO, USA) or Wako Pure Chemical Industries (Osaka, Japan).
Construction of adenoviral vectors
A cDNA encoding a phosphatidylinositol 5′-phosphatase-defective mutant of SHIP2 (ΔIP-SHIP2) containing Pro687 to Ala, Asp691 to Ala, and Arg692 to Gly changes was subcloned into the vector pAxCAwt, and transferred to recombinant adenovirus by homologous recombination utilizing an Adenovirus Expression Vector Kit (Takara Biomedicals, Tokyo, Japan) as described previously . The adenoviral vector encoding IRS-1 was also 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% fetal bovine serum (FBS), 250 nmol/l dexamethasone, 0.5 mmol/l isobutyl methylxanthine, and 500 nmol/l insulin. After 3 days, the differentiation medium was replaced with postdifferentiation medium containing 10% FBS and 500 nmol/l insulin. After 3 more days, the postdifferentiation medium was replaced with DMEM supplemented with 10% FBS. ΔIP-SHIP2 and IRS-1 were transiently expressed in differentiated 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. A multiplicity of infection (m.o.i.) of 10–40 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 prior to removal. Subsequent experiments were conducted 24–48 h after initial addition of the virus . The efficiency of adenovirus-mediated gene transfer of ΔIP-SHIP2 and IRS-1 was approximately 95%.
Measurements of PI(3,4,5)P3 and PI(3,4)P2 levels in vivo
The same numbers of 3T3-L1 adipocytes transfected with LacZ or ΔIP-SHIP2 were starved of phosphate overnight in phosphate-free DMEM (Life Technology), then starved of serum for 3 h. [32P]Orthophosphate (3.7 MBq/ml) was added, and the cells were cultured for an additional 2 h. Following the labelling period, the cells were incubated with or without 1 μmol/l insulin for 15 min. The reaction was terminated by washing once with ice-cold PBS, followed by the addition of methanol and 1 N HCl (1:1). The labelling of the cells with [32P]orthophosphate was conducted at the same time in both sets of transfected cells. Phospholipids were then extracted with chloroform. The extracted lipid was deacylated and subjected to amino-exchange high-performance liquid chromatography (HPLC) using a Partisphere strong anion-exchange column (Whatman) as described previously . The PI(3,4)P2 and PI(3,4,5)P3 levels in the same sample for each line were measured within a single HPLC run. The radioactivity was detected with an online radiochemical detector.
Chronic insulin treatment
3T3-L1 adipocytes grown in 6-well multiplates were incubated with DMEM containing 0.1% FBS with or without 100 nmol/l insulin at 37°C for various periods. For experiments with rapamycin treatment, 20 nmol/l rapamycin was added for 30 min before the addition of insulin. At the end of the chronic treatment with insulin, the cells were washed with PBS, incubated in serum-free DMEM for 30 min, and washed again with PBS. The cells were then treated with or without 17 nmol/l insulin for 5 min.
Plasma membrane fractionation
The cells were washed twice with PBS and once with HES buffer (255 mmol/l sucrose, 20 mmol/l HEPES, 1 mmol/l EDTA, 1 mmol/l phenylmethylsulphonyl fluoride [PMSF], 1 mmol/l Na3VO4, 2 μg/ml of aprotinin, and 50 ng/ml of 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 the plasma membrane (PM) . In brief, the homogenates were centrifuged at 19,000 g for 20 min. The pellet obtained from the spin was resuspended in HES buffer, layered onto a 1.12 mol/l sucrose cushion, and centrifuged at 100,000 g in a swing rotor for 60 min. A white fluffy band at the interface was collected, resuspended in HES buffer, and centrifuged at 40,000 g for 20 min, yielding a pellet of PM. All fractions 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.
Immunoprecipitation and western blotting
The cells or the plasma membrane preparation were lysed in a buffer containing 20 mmol/l Tris, 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 2.5 mmol/l sodium deoxycholate, 1 mmol/l β-glycerophosphate, 1% Triton X-100, 1 mmol/l PMSF, 1 mmol/l Na3VO4, 50 mmol/l sodium fluoride, 10 μg/ml of aprotinin, and 10 μmol/l 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 (PVDM) using a Bio-Rad Transblot apparatus. The membranes were blocked in a buffer containing 50 mmol/l Tris, 150 mmol/l NaCl, 0.1% Tween 20, and 2.5% bovine serum albumin (BSA) or 5% non-fat 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 mmol/l Tris, 150 mmol/l NaCl, and 0.1% Tween 20, pH 7.5, 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 (Amersham) . In each experiment, the intensity of the band derived from control cells was assigned a value of 1 arbitrary unit, and the intensity of all treated groups was expressed as a fold value of control.
Measurement of PKCλ activity
The cells were washed with ice-cold PBS and lysed with PKCλ buffer containing 50 mmol/l MOPS–HCl, 0.5% Triton X-100, 10% glycerol, 5 mmol/l EDTA, 5 mmol/l EGTA, 20 mmol/l NaF, 50 mmol/l β-glycerophosphate, 2 mmol/l Na3VO4, 2 mmol/l DTT, 1 μg/ml of leupeptin, and 2 mmol/l PMSF, pH 7.5. The lysates were centrifuged at 15,000 g for 20 min. The protein concentration in the resulting supernatants was determined with the use of bicinchoninic acid protein assay reagent (Pierce), and equal amounts of protein were subjected to immunoprecipitation with anti-PKCλ antibody. The immunoprecipitates were washed twice with PKCλ buffer containing 0.1% BSA, once with PKCλ buffer containing 0.1% BSA and 1 mol/l NaCl, and once with a solution containing 20 mmol/l Tris–HCl, 10% glycerol, 0.5 mmol/l EDTA, 0.5 mmol/l EGTA, 20 mmol/l 2-mercaptoethanol, 10 μg/ml of leupeptin, and 2 mmol/l PMSF, pH 7.5. Then, the precipitates were incubated for 14 min at 30°C with 14.8 kBq of [γ-32ATP] in a reaction mixture (25 μl) containing 35 mmol/l Tris, pH 7.5, 10 mmol/l MgCl2, 0.5 mmol/l EGTA, 0.1 mmol/l CaCl2, 40 μmol/l unlabelled ATP, 100 μg/ml of phosphatidylserine, and 30 μmol/l myelin basic protein (MBP) as a substrate. Kinase reactions were terminated by the addition of SDS sample buffer, and the samples were then fractionated by SDS-PAGE [8, 22]. The radioactivity incorporated into substrates was determined with a Fuji BAS 2000 image analyser.
Measurement of 2-DOG uptake
3T3-L1 adipocytes grown in 6-well multiplates were serum-starved for 3 h. The cells were treated with or without rapamycin for 30 min and further incubated with 17 nmol/l insulin for 6 h. The cells were washed once with PBS, three times with Krebs–Ringer phosphate (KRP)–HEPES buffer, 10 nmol/l HEPES, 131.2 mmol/l NaCl, 4.7 mmol/l KCl, 1.2 mmol/l MgSO4, 2.5 mmol/l CaCl2, and 2.5 mmol/l NaH2PO4, pH 6.0, and once with KRP–HEPES buffer containing 1% BSA, pH 7.4. The cells were then incubated with the same KRP–HEPES buffer for 1 h at 37°C. The cells were subsequently stimulated with various concentrations of insulin. Following 15 min of insulin treatment, 3.7 kBq of 2-[3H]DOG was added for 4 min. The reaction was stopped by the addition of 10 μmol/l cytochalasin B. The cells were washed three times with PBS and solubilized with 0.2 mmol/l SDS–0.2 N NaOH . The radioactivity incorporated into the cells was measured by liquid scintillation counting.
The data are represented as means ± SEM. p Values were determined using Student’s t test, and p<0.05 was considered statistically significant.
Effect of chronic insulin treatment on IRS-1 and SHIP2
Inhibition of endogenous SHIP2 function by expression of ΔIP-SHIP2
Effect of ΔIP-SHIP2 and IRS-1 expression, and pretreatment with rapamycin on insulin-induced phosphorylation of Akt after chronic insulin treatment
Effect of ΔIP-SHIP2 and IRS-1 expression, and pretreatment with rapamycin, on insulin-induced activation of PKCλ after chronic insulin treatment
Effect of ΔIP-SHIP2 and IRS-1 expression, and pretreatment with rapamycin, on insulin-induced 2-DOG uptake after chronic insulin treatment
Chronic insulin exposure is known to cause a subsequent insulin resistance, by reducing the level of IRS-1 via PI3-kinase and rapamycin-dependent pathways [17, 18, 19, 20, 21, 30, 31]. In fact, our results demonstrated that chronic insulin treatment induced a reduction in IRS-1 levels in a time-dependent manner. In addition to the impaired IRS-1–dependent signalling pathway, the present study showed increased amounts of SHIP2 following chronic insulin exposure. Since SHIP2 is the physiologically important negative regulator of insulin signalling with a fundamental impact on the state of insulin resistance [8, 9, 10, 11, 12], the increase in SHIP2 protein appears to be part of the novel molecular mechanism of insulin resistance caused by chronic insulin treatment. Because SHIP2 is translocated to the plasma membrane where it functions to hydrolyse PI(3,4,5)P3, the increase in the amount of SHIP2 protein in the plasma membrane preparation further supports the possible involvement of SHIP2 in insulin resistance in 3T3-L1 adipocytes .
We employed two approaches to ameliorate the decrease in IRS-1 levels caused by the chronic insulin treatment. As shown in Fig. 3, pretreatment with rapamycin prevented the mTOR-dependent proteosomal degradation of IRS-1 caused by the chronic insulin treatment. Overexpression of exogenous IRS-1 in advance normalized the decreased IRS-1 levels caused by the insulin treatment. On the other hand, endogenous SHIP2 function was efficiently inhibited by expression of the 5′-phosphatase defective dominant-negative SHIP2 (ΔIP-SHIP2) as shown in Fig. 2. These approaches would be useful for clarifying whether the rescue of insulin signalling at the level of IRS-1 and/or SHIP2 is effective in ameliorating insulin resistance caused by chronic insulin treatment. The decrease in the phosphorylation of Akt caused by the chronic insulin treatment was effectively ameliorated by either prevention of the decrease in IRS-1 by rapamycin treatment or advanced IRS-1 overexpression, or inhibition of endogenous SHIP2 function by expression of the dominant-negative SHIP2 (ΔIP-SHIP2). These results indicate that insulin-induced phosphorylation of Akt is closely associated with the IRS-1–mediated PI3-kinase pathway. In addition, the full input of insulin signal does not appear to be required for the sufficient phosphorylation of Akt, because amelioration of insulin signalling at the step involving IRS-1 or SHIP2 is sufficient for the efficient restoration of the phosphorylation of Akt.
Phosphorylation at both Thr308 and Ser473 is required for the full activation of Akt [8, 26, 27, 28]. In this context, our results showed that the rescue of IRS-1 levels by treatment with rapamycin and overexpression of IRS-1 in advance, and expression of the dominant-negative SHIP2 (ΔIP-SHIP2), efficiently ameliorated the decreased insulin-induced phosphorylation of Akt at both residues caused by the chronic insulin treatment. In contrast to the effective recovery of acute insulin stimulation of Akt phosphorylation, the recovery of acute insulin stimulation of PKCλ activation was only partial for both the restoration of IRS-1 levels and inhibition of SHIP2 function. Thus, pretreatment with rapamycin, advanced IRS-1 overexpression, and ΔIP-SHIP2 expression only partially ameliorated the insulin-induced activation of PKCλ to 49.8±3.8%, 67.2±8.5%, and 65.0±7.5%, respectively, of the control value. The rescue of the PKCλ activity was still partial with a combination of ΔIP-SHIP2 expression and rapamycin pretreatment (or IRS-1 expression—data not shown). Therefore, the insulin resistance caused by chronic treatment may also impair insulin signalling at the step important for PKCλ activation more directly in addition to the IRS-1–PI3-kinase pathway. It is possible that another insulin signalling system important for glucose uptake including the CAP–Cbl–TC10 pathway may be a candidate implicated in the signalling step, although further investigation is needed to clarify the issue [32, 33]. We can not rule out the possibility that full activation of the IRS-1–PI3-kinase pathway is required for the efficient activation of PKCλ, although resistance at the levels of IRS-1 and SHIP2 appears to be efficiently rescued by pretreatment with rapamycin and expression of ΔIP-SHIP2.
Interestingly, the decreased stimulation of 2-DOG uptake caused by chronic insulin treatment was only partly restored by the maintenance of IRS-1 levels by pretreatment with rapamycin or advanced overexpression of IRS-1, or expression of ΔIP-SHIP2 as shown in Fig. 5. These findings are consistent with the results of PKCλ activation, and not Akt activation. Although Akt and atypical PKC are downstream effectors of PI3-kinase strongly implicated in the metabolic actions of insulin, the relative importance of Akt versus atypical PKC in insulin-induced 2-DOG uptake is controversial [8, 22, 27, 28, 29, 34]. Our results indicate that PKCλ/ζ rather than Akt may be more closely linked to the insulin-stimulated glucose uptake and associated with the state of insulin resistance. It is unclear whether this difference between Akt and PKCλ activation in chronic insulin treatment reflects a small input of IRS-1–dependent insulin signalling sufficient for Akt activation, or whether factors other than IRS-1–dependent insulin signalling are involved in the impairment of PKCλ activation. In any event, our results indicate that PKCλ activity rather than Akt activity appears to be associated with the decreased glucose uptake caused by chronic insulin treatment. Regardless of the importance of PKCλ to the state of insulin resistance, overexpression of the constitutively active form of PKCλ did not completely rescue the decreased insulin-stimulated glucose uptake caused by the chronic insulin treatment (data not shown). Based on this observation, chronic insulin treatment appears to cause insulin resistance at multiple signalling steps including a step distal to the PKCλ activation leading to the glucose uptake. It is also possible that chronic insulin treatment impairs the glucose uptake involved in the Glut4 translocation system independent of insulin signalling as previously reported for the insulin resistance caused by dexamethasone treatment in 3T3-L1 adipocytes .
In summary, SHIP2 appears to participate in insulin resistance, at least in part, caused by chronic insulin treatment in 3T3-L1 adipocytes. In addition, (1) impaired early insulin signalling occurring mainly at IRS-1 for the PI3-kinase activation, (2) impaired insulin signalling for PKCλ activation, and (3) impaired glucose transport system may also be involved in the insulin resistance caused by chronic insulin treatment. Furthermore, the present study indicates that the inhibition of endogenous SHIP2 appears to be effective at ameliorating the insulin signal in a state of insulin resistance, and that the activity of PKCλ rather than Akt may be more closely associated with the decreased 2-DOG uptake caused by the chronic insulin treatment in 3T3-L1 adipocytes. Taken together, inhibition of the endogenous level and/or function of SHIP2 would be an important therapeutic target of insulin resistance in type 2 diabetes.
This work was supported in part by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science. We thank Dr Wataru Ogawa (Kobe University, Japan) for kindly providing the anti-PKCλ antibody and Dr Kazuyuki Hiratani (Toyama Medical and Pharmaceutical University, Japan) for technical assistance. T. Sasaoka and K. Fukui contributed equally to this work.
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