Archives of Biochemistry and Biophysics 558 (2014) 42–50

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Archives of Biochemistry and Biophysics

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Insulin-like modulation of Akt/FoxO signaling by copper ionsis independent of insulin receptor

http://dx.doi.org/10.1016/j.abb.2014.06.0040003-9861/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Institute of Nutrition, Department of Nutrigenomics,Friedrich-Schiller Universität Jena, Dornburger Str. 29, D-07743 Jena, Germany. Fax:+49 3641 949752.

E-mail address: lars-oliver.klotz@uni-jena.de (L.-O. Klotz).1 I.H. and K.P. are equal first authors.2 Abbreviations used: IR, insulin receptor; PI3K, phosphoinositide 30-kinase; PTPas-

es, protein tyrosine phosphatases; GSK3, glycogen synthase kinase 3; FoxO, forkheadbox, class O; G6Pase, glucose 6-phosphatase; ROS, reactive oxygen species; RTK,receptor tyrosine kinases; IGF1R, insulin-like growth factor-1 receptor; DMEM,Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; HBSS, Hanks’ balanced saltsolution; HRP, horseradish peroxidase; SDS, sodium dodecyl sulfate; BCA, bicinchon-inic acid; Ins, insulin; IB, immunoblotting.

Ingrit Hamann a,1, Kerstin Petroll a,b,1, Larson Grimm a, Andrea Hartwig b, Lars-Oliver Klotz a,c,⇑a Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canadab Karlsruhe Institute of Technology, Karlsruhe, Germanyc Institute of Nutrition, Department of Nutrigenomics, Friedrich-Schiller Universität Jena, Germany

a r t i c l e i n f o

Article history:Received 28 February 2014and in revised form 19 May 2014Available online 13 June 2014

Keywords:Insulin signalingPTPase (protein tyrosine phosphatase)ROSCopper ionsHepatoma cellsLinsitinibAktFoxO

a b s t r a c t

Copper ions are known to induce insulin-like effects in various cell lines, stimulating the phosphoinositide30-kinase (PI3K)/Akt signaling cascade and leading to the phosphorylation of downstream targets, includingFoxO transcription factors. The aim of this work was to study the role of insulin- and IGF1-receptors (IR andIGF1R) in insulin-like signaling induced by copper in HepG2 human hepatoma cells. Cells were exposed toCu(II) at various concentrations for up to 60 min. While Akt and FoxO1a/FoxO3a were strongly phosphor-ylated in copper- and insulin-treated cells at all time points studied, only faint tyrosine phosphorylation ofIR/IGF1R was detected in cells exposed to Cu(II) by either immunoprecipitation/immunoblot or by immu-noblotting using phospho-specific antibodies, whereas insulin triggered strong phosphorylation at thesesites. Pharmacological inhibition of IR/IGF1R modestly attenuated Cu-induced Akt and FoxO phosphoryla-tion, whereas no attenuation of Cu-induced Akt activation was achieved by siRNA-mediated IR depletion.Cu(II)-induced FoxO1a nuclear exclusion was only slightly impaired by pharmacological inhibition of IR/IGF1R, whereas insulin-induced effects were blunted. In contrast, genistein, a broad-spectrum tyrosinekinase inhibitor, at concentrations not affecting IR/IGF1R, attenuated Cu(II)-induced Akt phosphorylation,pointing to the requirement of tyrosine kinases other than IR/IGF1R for Cu(II)-induced signaling.

� 2014 Elsevier Inc. All rights reserved.

Introduction 30-kinase (PI3K)-dependent phosphorylation and activation of the

Copper ions may interfere with crucial signaling pathways inmammalian cells, resulting in potentially adverse outcomes suchas altered gene expression and proliferation [1]. Exposure to cop-per ions has previously been demonstrated to modulate stress-responsive pathways, such as mitogen-activated protein kinasepathways, and to affect transcription factors such as AP-1 or NF-jB [2–4]. Likewise, insulin signaling in hepatoma cells was shownto be imitated by exposure to copper ions in the absence of insulin:Cu(II) elicited the stimulation of known signaling events down-stream of the insulin receptor (IR),2 e.g. the phosphoinositide

serine/threonine kinase Akt [5]. Moreover, exposure to Cu(II) causedphosphorylation of glycogen synthase kinase 3 (GSK3) as well as oftranscription factors of the forkhead box, class O (FoxO) family [6],both of which are known substrates of Akt. Akt-dependent phos-phorylation of FoxO proteins leads to their inactivation and nuclearexclusion [7], which was indeed observed in cells expressing EGFP-tagged FoxO1a exposed to Cu(II) [6]. Insulin triggers these sameeffects, leading to inactivation of FoxOs and attenuation of FoxO-dependent expression of genes, such as those of gluconeogenesisenzymes like the catalytic subunit of glucose 6-phosphatase(G6Pase) [8] or of plasma proteins like selenoprotein P [9,10] andthe major copper protein in human plasma, ceruloplasmin [11].

It is currently unclear how Cu(II) induces these described insu-lin-like signaling effects and what the molecular targets of copperions in cells are that result in the modulation of signaling events.The molecular targets would be of interest for a definition of themode of action of copper ions.

Several reasons point to the insulin receptor as an obviouspotential target: (i) copper ions stimulate insulin-like signaling(i.e. activation of PI3K/Akt to a comparable extent, followed bycomparable FoxO phosphorylation). (ii) Reactive oxygen species(ROS) and several stressful agents, such as quinones, alkylating

I. Hamann et al. / Archives of Biochemistry and Biophysics 558 (2014) 42–50 43

agents and ultraviolet radiation, have previously been shown totrigger activation of receptor tyrosine kinases (RTK) [12–18]. (iii)Copper ions are redox-active entities potentially triggering ROSformation that could elicit RTK activation [1]. (iv) Insulin receptoris a RTK whose activation may be modulated by ROS, such ashydrogen peroxide [19].

Therefore, we set out to investigate whether copper imitatesinsulin by acting on the insulin receptor (IR) and the related insu-lin-like growth factor-1 receptor (IGF1R), thereby causing stimula-tion of downstream signaling. We here demonstrate that copperions strongly stimulate insulin-like signaling in a fashion indepen-dent of IR and IGF1R.

Materials and methods

Reagents and plasmids

All chemicals were from Sigma–Aldrich (Oakville, ON, Canada),if not mentioned otherwise. The insulin receptor tyrosine kinaseinhibitor, linsitinib (OSI-906), was from Selleckchem (Burlington,ON, Canada) and the general tyrosine kinase inhibitor, genistein,was from LKT Laboratories (St. Paul, MN, USA). Inhibitors were heldas stock solutions in DMSO and diluted into serum-free cell culturemedia for use. The FoxO1a-EGFP expression plasmid [20] waskindly provided by Dr. Andreas Barthel (Endokrinologikum,Bochum, Germany).

Cell culture and fluorescence microscopy analyses

HepG2 human hepatoma cells were purchased from the Ger-man collection of microorganisms and cell cultures (DSMZ, Braun-schweig, Germany) and were held in Dulbecco’s modified Eagle’smedium (DMEM, with 4500 mg/l glucose and 2 mM glutamine,Sigma–Aldrich) supplemented with 10% (v/v) fetal calf serum(FCS) (PAA, Etobicoke, ON, Canada), 1% penicillin/streptomycin(Life Technologies, Burlington, ON, Canada) and 1% non-essentialamino acids (Sigma–Aldrich), at 37 �C in a humidified atmospherewith 5% (v/v) CO2.

Cell viability was assessed using neutral red uptake. HepG2 cellswere grown to 60–70% confluence in 24 well-plates, treated withcopper for 1 h, washed with PBS and subsequently held inserum-free medium for another 24 h. Cells were then incubatedfor 2 h with neutral red solution (Sigma–Aldrich; 4 ml of 3.3 g neu-tral red/l PBS in 100 ml serum-free DMEM). Cells were washedtwice with PBS, followed by extraction of neutral red from viablecells by incubation with an ethanol/water/acetic acid (50:49:1, v/v/v) solution under gentle shaking at room temperature for 2 h.The dye-containing solution was then centrifuged and the absor-bance of the cell-free supernatant was measured at 550 nm (with405 nm as reference).

For treatment of cells with copper ions or other agents, HepG2cells were grown to near confluence, held in serum-free mediumfor 24 h, followed by the respective treatment. If indicated, cellswere preincubated with an inhibitor (genistein or linsitinib) for60 min prior to the respective treatment with copper or insulin,which was in the continued presence of the inhibitor. DMSO wasused as vehicle control. For exposure to copper ions or insulin, cellswere washed once with PBS and incubated for 30–60 min in thepresence of various concentrations of Cu(II) sulfate or insulindiluted into Hanks’ balanced salt solution (HBSS, Sigma–Aldrich).For exposure to hydrogen peroxide, cells were washed once withPBS and incubated for 30 min in the presence of various concentra-tions of H2O2 in HBSS.

For analysis of FoxO1a-EGFP subcellular localization by fluores-cence microscopy, HepG2 cells were grown to approximately 60%confluence in 9 cm2 cell culture dishes and transfected with 3 lg

FoxO1a-EGFP expression plasmid in serum-free DMEM for 24 husing Nanofectin transfection reagent as described by the manu-facturer (PAA). Following transfection, cells were washed withPBS, then incubated in the presence of copper(II) sulfate or insulinfor 60 min. Where applicable, cells were incubated in the presenceof linsitinib as described above. Fluorescence microscopy of cellsexpressing EGFP-tagged FoxO1a was performed on an AxiovertObserver.A1 fluorescence microscope (Zeiss, Göttingen, Germany)coupled to an AxioCam MRm camera (Zeiss) using suitable filters.Analysis of EGFP-positive cells was done by counting and separat-ing cells into three categories with respect to the major localizationof FoxO1a-EGFP (nuclear, cytosolic or both nuclear/cytosolic). Foreach determination, approximately 200 cells were counted.

Western blotting

For analysis of IR, Akt, FoxO1a, FoxO3a and beta-actin levels ormodifications, cells were lysed in 2� SDS–PAGE buffer [125 mMTris/HCl, 4% (w/v) SDS, 20% (w/v) glycerol, 100 mM dithiothreitoland 0.02% (w/v) bromophenol blue, pH 6.8], followed by briefsonication. Samples were applied to SDS–polyacrylamide gels of10% (w/v) acrylamide, electrophoretically separated and blottedonto nitrocellulose membranes. Membranes were blocked in 5%non-fat dry milk in Tris–buffered saline containing 0.1% (v/v)Tween-20 (TBST) and probed with primary antibody overnight at4 �C, followed by washing, incubation with secondary antibody[horseradish peroxidase (HRP)-conjugated anti-rabbit IgG orHRP-coupled anti-mouse IgG, GE-Healthcare (Piscataway, USA)]and detection using chemiluminescent HRP substrate. The follow-ing primary antibodies were used: anti-phospho-IR-b/IGF1R-b(Y1150/1151)/(Y1135/1136), anti-total-IR-b, anti-phospho-Akt(Ser473), anti-total-Akt, anti-phospho-FoxO1a/FoxO3a (T24/T32),anti total FoxO1a (all from Cell Signaling Technology, Danvers,MA, USA), anti-b-actin (Sigma–Aldrich) and GAPDH (Millipore,Billerica, MA, USA). All primary antibody incubations were in 5%(w/v) BSA in TBST, and all secondary antibody incubations werein 5% (w/v) non-fat dry milk in TBST. General tyrosine phosphoryla-tion was detected using a mouse monoclonal anti-phosphotyrosineantibody, ‘‘4G10 Platinum’’, which is a mixture of two anti-pYantibody clones, 4G10 and PY20 (Millipore).

Test for protein tyrosine phosphatase (PTPase) inhibition

Tyrosine phosphorylation of IR/IGF1R was stimulated by incu-bation of cells with insulin (100 nM) for 30 min. Insulin treatmentwas in the absence or presence of the known phosphatase inhibi-tor, vanadate (sodium orthovanadate at a final concentration of1 mM) or the compound(s) of interest whose PTPase inhibitoryactivity was being investigated. To prevent any further autophos-phorylation, cells were treated with 10 lM linsitinib. After 5 min,medium was quickly removed, cells washed with PBS and lysedin 2� SDS–PAGE sample buffer, followed by detection of phos-pho-IR-b/IGF1R-b (Y1150/1151)/(Y1135/1136) and b-actin byWestern blotting.

Immunoprecipitation

For immunoprecipitation, cells were grown to 70% confluencein 58 cm2 culture dishes as described above. After treatment, cellswere washed once with PBS and lysed in 500 ll RIPA buffer (1%IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS), 150 mM NaCl, 50 mM Tris–HCl (pH 8), 5 mM sodiumfluoride, 1 mM sodium vanadate, 1 mM b-glycerophosphate,2.5 mM sodium pyrophosphate, 1 lg/ml aprotinin, 1 mM phenyl-methylsulfonyl fluoride, 1 mM EDTA and 1 mM DTT), followed bybrief sonication. Insoluble material was removed by centrifugation

44 I. Hamann et al. / Archives of Biochemistry and Biophysics 558 (2014) 42–50

for 10 min at 14,000g and 4 �C. Protein concentration in superna-tants was determined using the bicinchoninic acid (BCA) assay(Thermo Fisher Scientific, Rockford, IL, USA), and equal amountsof protein (between 500 and 900 lg, depending on the experi-ment) from each lysate were incubated with 2 lg of precipitatingantibody overnight at 4 �C, as indicated in the respective figures[rabbit monoclonal anti-IR-b (4B8; Cell Signaling) or rabbit poly-clonal anti-IR-b (cat # sc-711; Santa Cruz Biotechnology, SantaCruz, CA, USA)]. Immune complexes were precipitated with proteinG magnetic beads (Life Technologies) or protein A/G agarose beads(Santa Cruz Biotechnology), separated from the lysate and washedthree times in RIPA buffer. Magnetic or agarose beads were resus-pended in 2� SDS–PAGE buffer, heat-denatured, centrifuged andsupernatants separated by SDS–PAGE on a 10% polyacrylamidegel, transferred to nitrocellulose membranes and analyzed for tyro-sine phosphorylation using the ‘‘4G10 Platinum’’ monoclonal anti-phosphotyrosine antibody. Immunoprecipitation was controlledfor by reprobing membranes with anti-IR-b (monoclonal or poly-clonal). Membrane blocking was in 5% (w/v) BSA in TBST, all pri-mary antibody incubations were in 1% (w/v) BSA in TBST and allsecondary antibody incubations were in TBST.

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Transfection of cells with siRNA for insulin receptor silencing

100 ll transfection mix were added to each well of a 24 wellplate. Transfection mix consisted of serum-free cell culture med-ium (DMEM as described above), 3 ll Dharmafect transfectionreagent 4 (Thermo Fisher Scientific, Rockford, IL, USA) and 50 nMSMARTpool ON-TARGETplus INSR siRNA (set of 4 sequences:GAACAAGGCUCCCGAGAGU, AAACGAGGCCCGAAGAUUU, ACGGA-GACCUGAAGAGCUA, GCAGGUCCCUUGGCGAUGU) or 50 nM ON-TARGETplus non-targeting siRNA pool (set of 4 sequences:UGGUUUACAUGUCGACUAA, UGGUUUACAUGUUGUGUGA, UGGUUUACAUGUUUUCUGA, UGGUUUACAUGUUUUCCUA) (all fromThermo Fisher Scientific). Trypsinized HepG2 cells were dilutedin 400 ll DMEM cell culture medium containing 10% FBS andadded to each well, yielding a cell confluence of 25%. 48 h post-transfection, cells were washed with PBS and treated with 10 or100 lM CuSO4 or 100 nM insulin in HBSS buffer for 60 min. Cellswere washed with PBS and lysed in 0.5% SDS. Following proteindetermination, lysates were mixed with 4� SDS–PAGE sample buf-fer, followed by detection of total InsR, phospho-IR-b/IGF1R-b(Y1150/1151)/(Y1135/1136), phospho-Akt (Ser473), anti-phospho-FoxO1a/FoxO3a (T24/T32) and GAPDH by Western blotting.

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Fig. 1. Exposure of hepatoma cells to copper: effect on insulin/IGF1 receptor andAkt activation. HepG2 human hepatoma cells were grown to near confluence,washed with PBS and exposed to copper sulfate (3–100 lM in HBSS) for 60 min.After incubation, cells were washed, lysed and analyzed for tyrosine phosphory-lation of insulin receptor (IR) and/or insulin-like growth factor-1 (IGF1) receptorand for phosphorylation of Akt by Western blotting and immunodetectionemploying phospho-specific antibodies. Insulin (100 nM in HBSS, 60 min) servedas a positive control. (A) IR phosphorylation at Tyr-1150/1151 and IGF1 receptorphosphorylation at Tyr-1135/1136, as well as total levels of IR and beta actin (as aloading control, not shown) were detected by immunoblotting and signal inten-sities assessed densitometrically. Phosphorylated receptor/total IR ratios wererelated to those of the respective control treatments (Ctrl) which were set equal to1. (B) Phosphorylation of Akt at Ser-473, total levels of Akt and beta actin (as aloading control, not shown) were assessed densitometrically and phospho-Akt/totalAkt ratios were related to those of the respective control treatments (Ctrl) whichhad been set equal to 1. (B, inset) logarithmic presentation of same data. The datashown are means of three independent experiments (±SEM).

Results and discussion

Insulin-mimetic signaling induced by copper ions: comparison of Aktand insulin receptor phosphorylation

Copper ions were previously demonstrated to strongly stimu-late the Ser/Thr kinase Akt in a phosphoinositide 30-kinase(PI3K)-dependent fashion [5]. In order to investigate whether thiseffect initiates at the level of the insulin receptor (IR) or the relatedinsulin-like growth factor receptor-1 (IGF1R), we tested for a stim-ulation of IR and IGF1R tyrosine phosphorylation using phospho-specific antibodies. The efficacy of the copper exposure conditionschosen was previously established by us in the same cell culturemodel employed here [21]. Copper concentrations chosen werebetween 3 and 100 lM, and while there was no detectable lossin cell viability even 24 h after a 1 h exposure to 10 lM Cu(II),viabilities were lower with 30 lM and 100 lM (with 60% and20% viability 24 h after exposure, respectively; data not shown).

Using a phospho-specific antibody, we analyzed an IR tyrosineresidue cluster required to be phosphorylated for full activation

of the receptor upon stimulation and that contains Tyr-1150 andTyr-1151 (numbers referring to the short, IR-A isoform, corre-sponding to tyrosines 1162/1163 in IR-B). These sites correspondto Tyr-1135/Tyr-1136 in IGF1R, whose phosphorylation would bedetected by the same antibody. Stimulation of HepG2 cells withinsulin induced a strong phosphorylation of these tyrosines,whereas copper elicited a slight increase in tyrosine phosphoryla-tion (between approx. 1.5 and 2.5-fold over control; Fig. 1A). This isin stark contrast, however, to the observed strong Akt phosphory-lation at Ser-473 in cells exposed to copper ions, which starts atless than 10 lM and even exceeds the effect elicited by insulin(Fig. 1B). Neither a 5, 30 nor 60 min exposure to copper ions elic-ited significant IR/IGF1R phosphorylation (i.e. no more than inFig. 1A), whereas there was (i) a distinct activation of Akt by copperions and (ii) a strong stimulation of IR phosphorylation by insulinat all times (data not shown).

In order to test whether any other insulin receptor tyrosine res-idues might be phosphorylated upon exposure of cells to copperions, we performed an immunoprecipitation of the insulin recep-tor, followed by Western blotting analysis of general tyrosinephosphorylation with an anti-phospho-tyrosine antibody. No cop-per-induced tyrosine phosphorylation of the insulin receptor wasdetectable, whereas insulin expectedly caused a significanttyrosine phosphorylation of its receptor (Fig. 2A). In order to fur-ther substantiate these data, we also used a polyclonal antibody

IP: IR polyclonalIB: pTyr

IB: IR polyclonal

IP: IR monoclonal

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Fig. 2. Effect of copper on insulin receptor tyrosine phosphorylation in HepG2 cells.HepG2 human hepatoma cells were grown to near confluence, washed with PBSand exposed to copper (10, 30, 100 lM in HBSS) for 60 min. Insulin (100 nM inHBSS) served as a positive control. After incubation, cells were washed with PBS,solubilized in RIPA buffer and subjected to immunoprecipitation (IP) followed byimmunoblotting. All blots are representative of at least three independentexperiments. (A) IR-b (4B8) (monoclonal) immunoprecipitates were furtheranalyzed by Western blotting using anti-phosphotyrosine and anti-IR-b (monoclo-nal) antibodies. (B) IR-b (polyclonal) (C19) immunoprecipitates were furtheranalyzed by Western blotting using anti-phosphotyrosine and anti-IR-b (poly-clonal) antibodies. Tyrosine phosphorylation of IR-b and total levels of IR-b (asloading control) were assessed densitometrically and the phospho-IR b/total IRratios were related to those of the respective control treatments (Ctrl) which wasset equal to 1. The graph shows means of three independent experiments (±SEM).

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Fig. 3. Effect of IR inhibitor on IR and Akt phosphorylation in cells exposed tocopper. HepG2 human hepatoma cells were grown to near confluence, washed withPBS and exposed to copper (10 and 100 lM in HBSS) or insulin (100 nM in HBSS) for60 min. Where indicated, the insulin receptor tyrosine kinase inhibitor, linsitinib(OSI-906), was added to culture media to a final concentration of 1 lM 60 min priorto insulin or copper treatment and was further present throughout treatment.DMSO was used as vehicle control (‘‘�’’). After incubation, cells were washed, lysedand analyzed for phosphorylation and level of IR (A) and Akt (B) by Westernblotting and immunodetection employing phospho-specific antibodies as describedin the legend to Fig. 1. All phospho-protein/total protein ratios were related to thoseof the respective control/DMSO treatments (Ctrl/�) which was set equal to 1. (B,inset) Presentation of copper data (10 and 100 lM) from four individual experi-ments to illustrate inhibitor effect at lower copper concentrations. Copper-inducedAkt phosphorylation in the absence of inhibitor was set equal to 1 in eachexperiment. Data shown in the bar graphs result from densitometric analyses offour independent experiments (±SEM).

I. Hamann et al. / Archives of Biochemistry and Biophysics 558 (2014) 42–50 45

for precipitation of IR, followed by phospho-tyrosine detection –with a slightly different result: a trend toward tyrosine phosphor-ylation of the IR was observed in cells exposed to copper ions.Insulin-induced tyrosine phosphorylation of its receptor againwas far more substantial than the effect elicited by copper ions(Fig. 2B).

Copper-induced signaling: role of insulin receptor

Although copper only modestly elevated tyrosine phosphoryla-tion (and activity) of the IR, it cannot be excluded that this minorincrease in IR activity is required and sufficient for initiation ofCu signaling. We therefore employed linsitinib (OSI-906), a dualIR/IGF1R inhibitor [22], to test for the role of IR/IGF1R in Cu-induced insulin-like signaling. The concentration of linsitinib thatwas used (1 lM) was sufficient to fully abrogate both basal andinsulin-induced tyrosine phosphorylation of the IR/IGF1R(Fig. 3A). Likewise, insulin-induced Akt activation was blunted inthe presence of linsitinib, whereas copper-induced Akt activationwas only partly attenuated (Fig. 3B).

The above-mentioned slight stimulation of IR/IGF1R tyrosinephosphorylation is again seen in Fig. 3A.

Interestingly, whereas the effect on Akt of 100 lM Cu2+ was notsignificantly blocked by linsitinib, a clear inhibitory effect of thecompound on copper-induced Akt activation was detected at10 lM of Cu2+ in 2 of 4 independent experiments (Fig. 3B, inset).

Akt phosphorylates and inactivates transcription factors of theFoxO family, which were previously shown to be phosphorylatedand inactivated in cells exposed to copper ions [1,6]. Fig. 4A dem-onstrates that copper ions induce a strong phosphorylation ofFoxO1a and FoxO3a at a site known to be phosphorylated by Akt(Thr-24/Thr-32). At 10 lM Cu2+, this phosphorylation is as strongas the one induced by insulin. Inhibition of IR/IGF1R activity using

linsitinib did not impair copper-induced phosphorylation at100 lM Cu2+, and no more than an inhibitory trend was observedat 10 lM Cu2+; in contrast, insulin-induced FoxO phosphorylationwas back to control levels in the presence of the inhibitor (Fig. 4B).

In order to test whether this finding regarding the role of theinsulin receptor in FoxO phosphorylation also translates tofunctional consequences, we tested for alterations in FoxO1asubcellular localization.

HepG2 human hepatoma cells were transiently transfectedwith a plasmid coding for an EGFP-tagged version of FoxO1a.Transfected cells were pretreated with linsitinib or vehicle control(DMSO), followed by addition of insulin/copper in the continuedpresence of the inhibitor/DMSO. Subcellular localization ofFoxO1a-EGFP was then analyzed microscopically.

Under basal conditions, FoxO1a-EGFP was predominantlynuclear in roughly 10–15% of all cells analyzed, whereas less than10% had the protein exclusively cytosolic. Approximately 80% ofthe cells had both nuclear and cytoplasmic FoxO1a-EGFP. Thenumbers of cells with nuclear and cytosolic FoxO1a-EGFP wereset equal to 1 for control conditions and changes detected uponexposure to copper or insulin were related to these numbers.

As expected, and in line with causing Akt-dependent FoxOphosphorylation, insulin stimulated nuclear exclusion of FoxO1aproteins, resulting in a decrease in relative numbers of cells carry-ing FoxO1a-EGFP predominantly in the nucleus (black bars) and an

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Fig. 4. Effect of copper on Forkhead box, class O (FoxO) phosphorylation – effect oflinsitinib. (A) HepG2 cells were grown to near confluence, washed with PBS andexposed to copper (3–100 lM in HBSS) for 60 min. After incubation, cells werewashed, lysed and analyzed for phosphorylation of FoxO proteins by Westernblotting and immunodetection employing phospho-specific antibodies. Insulin(100 nM in HBSS) served as the positive control. Phosphorylation at Thr 24 (FoxO1a)or Thr 32 (FoxO3a), total levels of FoxO1a and of beta-actin (as a loading control, notshown) were assessed densitometrically and phospho-FoxO1a and 3a/total FoxO1aratios were related to those of the respective control treatments (Ctrl) which wasset equal to 1. The graph shows means of three independent experiments (±SEM).(B) HepG2 cells grown to near confluence were washed with PBS and exposed tocopper (10 and 100 lM in HBSS) for 60 min. Insulin (100 nM in HBSS) served as apositive control. Where indicated, the insulin receptor tyrosine kinase inhibitor,linsitinib (OSI-906), was added to culture media to a final concentration of 1 lM60 min prior to insulin or copper treatment and was present throughout. DMSO wasused as vehicle control (‘‘�’’). After incubation, cells were washed, lysed andanalyzed for phosphorylation and level of FoxO1a/3a employing phospho-specificantibodies as described in (A). Data shown in the bar graphs are means of fourindependent experiments (±SEM).

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Fig. 5. Copper-induced modulation of FoxO1a-EGFP subcellular localization: effectof linsitinib. HepG2 human hepatoma cells were transfected with a FoxO1a-EGFPexpression plasmid. After 24 h, insulin (100 nM in HBSS) or copper (10 or 100 lM inHBSS) were added to the cultures for 60 min, followed by fluorescence microscopy.Where indicated, the insulin receptor tyrosine kinase inhibitor, linsitinib (OSI-906),was added to culture media to a final concentration of 1 lM 60 min prior to insulinor copper treatment and was present throughout. DMSO was used as a vehiclecontrol. Approx. 200 EGFP-positive cells were counted for cell classification withrespect to predominant localization of EGFP. Data are means of three independentexperiments (±SEM).

46 I. Hamann et al. / Archives of Biochemistry and Biophysics 558 (2014) 42–50

increase in numbers of cells with cytosolic FoxO1a-EGFP (graybars; Fig. 5). Cu at 100 lM even more potently induced FoxO1a-EGFP nuclear exclusion, thus imitating insulin. This effect wasmuch less intense at 10 lM Cu, but a trend to nuclear exclusionwas still observed.

While insulin-induced nuclear exclusion was largely preventedin the presence of linsitinib, there was no effect of the inhibitor onCu (100 lM)-induced nuclear exclusion, implying that this coppereffect is independent of IR/IGF1R. The inhibitor effect on Cu(10 lM)-induced nuclear exclusion was minor (Fig. 5).

In addition to using the selective IR/IGF1R inhibitor linsitinib toanalyze whether IR phosphorylation and activity are involved in Cusignaling, we used siRNA to knock down endogenous IR. HepG2cells were transiently transfected with a mixture of four differentsiRNAs targeting the IR, achieving a downregulation of IR proteinlevels of about 50–60% (see Fig. 6A and C, detection of IR). Asshown in Fig. 6, insulin induced strong phosphorylation of IR/IGF1R(Fig. 6A), of Akt and of FoxO (Fig. 6B) in cells transfected with con-trol (i.e., non-depleting) siRNA, whereas IR-specific siRNA effi-ciently attenuated insulin-induced phosphorylation of theseproteins (see Fig. 6C, white vs. black bars).

In sharp contrast, IR knockdown in HepG2 cells did not affectCu-induced Akt and FoxO phosphorylation, neither at 10 nor100 lM (Fig. 6B and C, gray vs. black bars).

In summary, copper ions only very modestly stimulate IR/IGF1Rphosphorylation in HepG2 cells, and experiments using a pharma-cological inhibitor or an siRNA-based approach suggest that thestrong stimulation of Akt/FoxO signaling by Cu2+ is largely inde-pendent of IR/IGF1R stimulation.

The inhibitory action of linsitinib was seen exclusively at lower,non-cytotoxic copper concentrations, suggesting that there is onlya minor contribution of IR/IGF1R to Cu-induced Akt signaling, andthat with increasing Cu concentrations this contribution is over-ruled by some copper effect that is yet to be defined. In supportof this hypothesis, there was no concentration-dependent increasein the slight Cu-induced IR phosphorylation observed (Figs. 1A, 2Band 3A), whereas such concentration-dependent increase in phos-phorylation was obvious for Akt (Fig. 1B) and FoxOs (Fig. 4A).Hence, increasing Cu concentrations would stimulate an effectother than IR/IGF1R activation, resulting in Akt signaling on topof the effects on Akt caused by IR/IGF1R. The concept of a minoror basal activity of IR/IGF1R being required for one layer of Akt sig-naling whereas contributors other than IR/IGF1R cause theobserved strong activation of Akt is in line with our findings inthe siRNA experiments. Even after lowering IR levels using siRNA,residual IR levels remained – likely sufficient for basal low IR activ-ity, which might suffice for Cu signaling: different from copper sig-naling, insulin signaling is strongly dependent on the presence ofIR, resulting in the observed attenuation of insulin effects on Akt(Fig. 6).

In order to test the hypothesis that copper ions initiate signalingthat also causes Akt activation independent of IR/IGF1R on top ofthe minor contributions of IR/IGF1R, we then tested whether

C IR C IR C IRC IR siRNAp-IR/IGF1R

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Fig. 6. Effect of IR silencing on IR, Akt and FoxO phosphorylation by copper ions. HepG2 cells were transfected with non-depleting control siRNA or IR-specific siRNA for 48 h,washed with PBS and exposed to copper (10, 100 lM) or 100 nM insulin (Ins) for 60 min in HBSS. Cells were washed in PBS and lysed in 0.5% SDS. After protein determinationlysates were mixed with 4� SDS–PAGE sample buffer, followed by Western blotting with detection of the amount of (A) total IR protein and InsR/IGF1 receptor, and (B) Aktand FoxO phosphorylation, as well as of GAPDH (loading control). Blots shown are representative of three independent experiments with similar results. (C) Quantitation ofsignal intensities; signals observed in samples from cells transfected with IR-specific siRNA were related to control siRNA data, which were set to 1. Data shown in the bargraphs result from densitometric analyses of three independent experiments (±SD).

I. Hamann et al. / Archives of Biochemistry and Biophysics 558 (2014) 42–50 47

tyrosine kinases other than the IR/IGF1R contribute to Cu-dependent Akt activation.

Copper-induced signaling: role of other tyrosine kinases?

As shown in Fig. 7A, an increased extent of general tyrosinephosphorylation of numerous proteins across a broad range ofmolecular masses is detectable in lysates of cells exposed to Cu(II),implying either a copper-induced tyrosine kinase activation ortyrosine phosphatase inactivation, resulting in a net increase intyrosine phosphorylation. In order to test whether the stimulationof tyrosine kinases other than IR/IGF1R may be instrumental in Aktactivation by copper at all, we used genistein, a general tyrosinekinase inhibitor [23] that, while affecting some insulin-inducedbiochemical processes, does not appear to potently block auto-phosphorylation of the insulin receptor itself [24]. At 40 lM, a con-centration sufficient to block epidermal growth factor-induced Aktactivation in HepG2 cells (data not shown and [25]), insulin-induced IR/IGF1R (Fig. 7B), Akt (Fig. 7C) or FoxO (Fig. 7D) phos-phorylation were not affected.

Despite insulin effects not being altered at this concentration,genistein attenuated Cu(II) (10 and 100 lM)-induced Akt and FoxO(at 10 lM Cu2+) phosphorylation (Fig. 7C/inset and D), implyingthat tyrosine kinase activity other than the intrinsic activity ofthe IR or IGF1R contribute to copper-induced activation of Akt.

In summary, Cu(II)-induced Akt activation is largely indepen-dent of IR/IGF1R, and occurs, at least in part, through protein tyro-sine kinases yet to be identified.

We then asked how copper can stimulate tyrosine phosphoryla-tion and tested for potential molecular mechanisms.

Copper-induced insulin signaling: inhibition of PTPases?

Metal ions, including copper ions, can affect cellular signalingcascades in at least two different ways – by stimulating the pro-duction of reactive oxygen species (ROS) or by directly binding toproteins involved in signaling (for review, see [26]). One group ofproteins that would be affected by both options are protein

tyrosine phosphatases (PTPases). PTPases catalyze thedephosphorylation of phosphotyrosyl residues of protein sub-strates and are known to be prone to inactivation both by ROS(such as hydrogen peroxide) or by interaction with copper ions,owing to an active site cysteine (see [1,27,28] for review). PTPaseinactivation could therefore result in a net enhancement of tyro-sine phosphorylation. We set out to test for a contribution ofhydrogen peroxide and of PTPases in our setting of copper-inducedinsulin-like signaling.

In order to test whether the formation of hydrogen peroxidein cells exposed to copper ions might be a likely mediator ofthe observed signaling effects, we exposed cells to hydrogen per-oxide. This approach is valid only under the assumption thathydrogen peroxide applied exogenously will reach the cell’sinterior. Indeed, hydrogen peroxide permeation of the plasmamembrane was found to be ‘‘limited’’ (see [29] for review) –but it is still significant and rapid, especially if facilitated by aqu-aporins such as AQP3 [30,31] and AQP8 [31,32], both of whichare expressed in HepG2 cells [33,34]. Interestingly, phosphoryla-tion of Akt and FoxO, but not the IR/IGF1R was detected in thesecells (Fig. 8). However, the concentrations of peroxide required toelicit Akt or FoxO phosphorylation in an extent comparable tothat elicited by 10 lM Cu(II) is in the 10 mM range. Even assum-ing a steep H2O2 concentration gradient across the HepG2 plasmamembrane, leading to an assumed intracellular H2O2 steady-stateconcentration in the high lM region, this would imply that thesehigh lM concentrations would have to be generated in cellsexposed to 10 lM Cu(II) (Fig. 8). A similar finding was reportedfor the epidermal growth factor receptor whose activation byhydrogen peroxide in fibroblasts required high millimolar H2O2

concentrations unless a sensitizing PTPase inhibitor, orthovana-date, was co-applied [17]. We therefore suggest that copper-induced ROS formation is not required for the induction of Aktand FoxO phosphorylation by Cu(II), which is in line with previ-ous findings in HeLa cells and fibroblasts that demonstrated theformation of ROS in cells exposed to copper ions and that thisROS formation observed slower kinetics than the phosphorylationof Akt [5,35].

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Fig. 7. Effect of genistein on IR and Akt activation by copper ions. (A) HepG2 cells were grown to near confluence, washed with PBS and exposed to copper (10, 30, 100 lM inHBSS) for 60 min. After incubation, cells were washed with PBS, solubilized in RIPA buffer and analyzed by immunoblotting (IB) using an anti-phosphotyrosine antibody. Theblot shown is representative of three independent experiments. (B–D) HepG2 cells were grown to near confluence, washed with PBS and exposed to copper (10 and 100 lM inHBSS) for 60 min. Insulin (100 nM in HBSS) served as positive control. Where indicated, the general tyrosine kinase inhibitor, genistein, was added to culture media to a finalconcentration of 40 lM 60 min prior to insulin or copper treatment and was present throughout. DMSO was used as a vehicle control (‘‘�’’). After incubation, cells werewashed, lysed and analyzed for phosphorylation and level of IR/IGF1R (B), Akt (C) and FoxO1a/3a (D) by Western blotting and immunodetection employing phospho-specificantibodies as described in the legend to Fig. 1. Phospho-protein/total protein ratios were related to those of the respective control/DMSO treatments (Ctrl/�) which were setequal to 1. Data shown in the bar graphs result from densitometric analyses of three independent experiments (±SEM).

48 I. Hamann et al. / Archives of Biochemistry and Biophysics 558 (2014) 42–50

We then devised an assay (Fig. 9A) to detect whether copperions interfere with regulatory circuits controlling IR/IGF1Rphosphorylation levels at all, i.e. whether tyrosine kinase activityof the IR/IGF1R or PTPases that regulate IR/IGF1R phosphorylationare in any way affected by copper ions. To that end, IR/IGF1Rtyrosine phosphorylation was stimulated by the addition of insu-lin. We then blocked IR/IGF1R tyrosine kinase activity using linsit-inib, and followed dephosphorylation of the receptor over time.After 5 min, dephosphorylation almost back to control conditionswas achieved (Fig. 9B, lane 3 versus lane 1), suggesting the pres-ence of a PTPase activity that controlled IR/IGF1R tyrosinephosphorylation.

The presence of a PTPase inhibitor should attenuate or evenblock dephosphorylation of IR/IGF1R. We chose orthovanadate, awell-known PTPase inhibitor, as positive control for the assay. Asdetected in lane 12 of Fig. 9B, tyrosine phosphorylation of IR/IGF1Rwas indeed upheld if insulin treatment was in the presence of van-adate, even if IR/IGF1R tyrosine kinase activity was blocked by lin-sitinib. In the presence of copper ions, a similar attenuation ofdephosphorylation was observed (Fig. 9B, lanes 6 and 9). Moreover,copper and vanadate ions seem to slightly enhance insulin-inducedIR/IGF1R phosphorylation (Fig. 9, lanes 5, 8 and 11; compare withlane 2 for control).

We also tested for Akt phosphorylation (at Ser-473) underthe same experimental conditions (Fig. 9B, row 2). As with IR/IGF1R tyrosine phosphorylation, Akt serine phosphorylationinduced by insulin exposure was almost entirely back to controlusing linsitinib (lane3). Similarly, Akt phosphorylation wasmaintained in the presence of vanadate – due to some tyrosinephosphatase upstream of Akt that was blocked by vanadate(lane 12). Regarding Cu(II), however, an interference with Aktdephosphorylation cannot be concluded from this experimentsince serine (rather than tyrosine) phosphorylation is the crucialactivating posttranslational modification of Akt and because, incontrast to vanadate, copper ions strongly induced Akt phos-phorylation per se (Fig. 9B, lanes 4 and 7; compare with lane10 for vanadate).

In summary, copper ions significantly impaired IR/IGF1Rdephosphorylation in HepG2 cells. Although this cannot fullyexplain Cu(II)-induced Akt signaling, simply because there is onlylimited contribution of IR/IGF1R to it, the observed impairmentof dephosphorylation provides a molecular mechanism for theminor contribution of IR/IGFR1 to Cu(II)-induced Akt signaling.As all known protein tyrosine phosphatases (PTPases) harbor verysimilar active sites (see also below), these data also imply that cop-per ions may impair PTPase activity in general.

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Fig. 8. Stimulation of insulin signaling: hydrogen peroxide versus copper ions. HepG2 human hepatoma cells were grown to near confluence, washed with PBS and exposed tohydrogen peroxide (1–100 mM in HBSS), copper sulfate (10 and 100 lM) or insulin (100 nM) for 30 min. After incubation, cells were washed, lysed and analyzed by Western blottingfor phosphorylation of insulin receptor (IR) and/or insulin-like growth factor-1 (IGF1) receptor as well as of Akt and FoxO1a/3a and for levels of beta actin (as loading control). Signalintensities were assessed densitometrically. Phosphorylated proteins/beta actin ratios were set equal to 1. The data are means of two independent experiments (±range/2).

+Ins

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Fig. 9. Analysis of PTPase inhibition by copper ions. (A) Schematic representation ofthe protein-tyrosine phosphatase (PTPase) inhibition assay. Insulin-induced auto-phosphorylation of insulin-receptor (IR) is blocked by linsitinib which preventsfurther phosphorylation. PTPase(s) deactivate(s) IR by dephosphorylating itstyrosines. The potent PTPase inhibitor vanadate inactivates PTPase(s) and therebyprevents dephosphorylation and inactivation of IR. (B) HepG2 cells were stimulatedwith insulin (100 nM) in the absence or presence of copper(II) sulfate (10 or100 lM) or sodium orthovanadate (1 mM) for 30 min in HBSS. Linsitinib (10 lM) orDMSO as vehicle control were added. After 5 min, medium was quickly removed;cells were washed with PBS and lysed in 2� SDS–PAGE sample buffer, followed byWestern blotting with detection of IR and IGF1 receptor phosphorylation at Tyr-1150/1151 and Tyr-1135/1136, respectively, as well as of phospho-Akt at Ser-473and GAPDH. Blots shown are representative of three independent experiments withsimilar results.

I. Hamann et al. / Archives of Biochemistry and Biophysics 558 (2014) 42–50 49

Conclusions

Copper ion-induced stimulation of insulin-like signaling inhuman hepatoma cells is demonstrated here to be initiated in a lar-gely insulin/IGF1 receptor-independent manner. Even at concentra-tions eliciting very strong stimulation of Akt, copper ions cause onlya minimal increase in IR tyrosine phosphorylation. Further, com-plete abrogation of IR or IGF1R activation moderately, but not fullyattenuates copper-induced Akt and FoxO phosphorylation.

Copper ions, therefore, imitate signaling effects of insulin, butthey do not mimic insulin’s mode of action, i.e. they do not fullystimulate its receptor. Although inducing no more than a modestIR/IGF1R tyrosine phosphorylation, copper ions are capable ofstimulating significant general tyrosine phosphorylation in cells.The fact that genistein attenuates Cu(II)-induced Akt activationpoints to a potential role of tyrosine kinases other than the IR/IGF1R in copper-induced Akt activation.

Although copper ions have recently been shown to bind to andstimulate a tyrosine kinase, the dual-specificity (i.e., a tyrosine-and Ser/Thr) kinase MEK-1 [36], we are not aware of otherexamples of copper binding stimulating (rather than inhibiting) atyrosine kinase. We therefore hypothesize that the effect of copperon overall tyrosine phosphorylation and, more specifically, on tyro-sine phosphorylation involved in Akt/FoxO signaling, is due toinactivation of (a) PTPase(s). It has been demonstrated previouslythat copper ions bind to, and inactivate PTPases, such as PTP1Band human vaccinia H1-related phosphatase (VHR) [1,37]. Mostimportantly, however, such an inhibition of a PTPase might explainthe broad spectrum of proteins whose tyrosine phosphorylationappears to be stimulated in the presence of copper (Fig. 7A): PTP-ases, while all sharing the active site cysteine thiolate that wouldallow for an inhibition by copper ions, tend to have multiple sub-strates. PTPase inhibition would therefore shift tyrosine phosphor-ylation/dephosphorylation equilibria to the dephosphorylation

50 I. Hamann et al. / Archives of Biochemistry and Biophysics 558 (2014) 42–50

side for multiple tyrosine kinase/substrate pairs. Obviously, theinduction of phosphorylation by inhibition of dephosphorylationrequires at least basal tyrosine kinase activity – which couldexplain the modest activation of the IR/IGF1R by copper and theinhibitory effect of genistein on Cu signaling.

Regarding the identity of such PTPases, we have observed a dis-tinct inhibitory effect of copper ions on PTPases directly regulatingthe IR/IGF1R in HepG2 cells (Fig. 9). As copper-induced modulationof Akt is independent of IR/IGF1R, it is unlikely that a PTPase reg-ulating these receptors is the major target causing Akt phosphory-lation. Yet PTEN, a PTPase-family phosphatase crucial to IR/Akt/FoxO signaling, but acting downstream of IR/IGF1R by dephospho-rylating phosphatidylinositol-30,40,50-trisphosphate, was recentlydemonstrated to be inhibited by Zn ions and shown to be requiredfor a Zn-induced modulation of Akt phosphorylation [38]. Based onthe similar coordination properties of Zn(II) and intracellular Cu(I)[1], we hypothesize that these findings may also apply to copper-induced Akt activation in HepG2 cells. In contrast to Zn ions, how-ever, copper ions are redox active in biological systems. Therefore,it remains to be elucidated both if copper ions inhibit PTEN inHepG2 cells at all and whether this is through coordinative occupa-tion or through oxidation of the phosphatase’s active site.

Acknowledgments

This study was supported by the Natural Sciences and Engineer-ing Research Council of Canada (NSERC, Discovery Grant RGPIN402228-2011 to LOK). LOK acknowledges support by Canada Foun-dation for Innovation (CFI), Alberta Advanced Education and Tech-nology (AET), and the Canada Research Chairs (CRC) Program.

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