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Redox-Dependent and Ligand-IndependentTrans-Activation of Insulin Receptor by Globular

AdiponectinTania Fiaschi, Francesca Buricchi, Giacomo Cozzi, Stephanie Matthias, Matteo Parri, Giovanni Raugei,

Giampietro Ramponi, and Paola Chiarugi

Adiponectin/ACRP30 is an adipose tissue–derived hormone with antiatherogenic, antidia-betic, and insulin-sensitizing properties. Although the metabolic effects of adiponectin onglucose and lipid metabolism are well known, the signaling pathways triggered by adiponec-tin receptors remain to be elucidated. We report evidence that in hepatic cells, adiponectinstimulation produces a transient burst of reactive oxygen species (ROS) through activationof the small GTPase Rac1 and 5-lypoxigenase. Furthermore, adiponectin-induced oxidantscause the oxidation/inhibition of protein-tyrosine phosphatase (PTP) 1B, one of the majorphosphotyrosine phosphatases involved in the control of insulin receptor phosphorylation.Adiponectin causes increased association of PTP1B to insulin receptor and the oxidation/inhibition of the phosphatase, ultimately provoking the ligand-independent trans-phospho-rylation of insulin receptor. We also report evidence that redox signaling plays a key role inboth mitogen-activated protein kinase activation and hepatic glucose consumption inducedby adiponectin. Conclusion: These results point to ROS as critical regulators of the cross-talkbetween adiponectin and insulin pathways and provide a redox-based molecular mechanismfor the insulin-sensitizing function of adiponectin. (HEPATOLOGY 2007;46:000-000.)

Adiponectin/ACRP30 is an abundant adipocyte-derived circulating plasma protein with insulin-sensitizing metabolic effects and vascular protective

properties.1 Adiponectin is composed of 2 structurallydistinct domains, an N-terminal collagen-like domainand a C-terminal globular domain. Both the full-length

and the globular form of the hormone have been observedin mammalian plasma.2 Furthermore, adiponectin mole-cules can associate in the plasma to form trimers, hexam-ers, and high molecular weight forms whose biologicalactivities are poorly understood.3 Studies in humans andmonkeys show that plasma adiponectin level significantlycorrelates with insulin sensitivity in the whole body.4,5

Moreover, the hormone treatment improves diabetes inmice.6 These findings identify adiponectin as an insulin-sensitizing hormone and indicate that its reduced produc-tion might be related to the pathophysiology of insulinresistance. The physiological effects of adiponectin onglucose and lipid metabolism in the liver and in skeletalmuscle are mediated by 2 receptors (AdipoR1 and Adi-poR2) that have been identified recently.7 These receptorscontain 7-transmembrane domains but are structurallyand functionally distinct from G-protein–coupled recep-tors. However, the signaling pathways responsible for themetabolic effects of adiponectin have been only partiallyelucidated. An involvement of AMP kinase (AMPK) ac-tivation has been indicated for both full-length and glob-ular adiponectin (gAd) in muscle and only for the full-length hormone in the liver, suggesting different organtargeting of the 2 adiponectin forms.7 In muscle, AMPKactivation has been correlated to fatty acid oxidation and

Abbreviations: 5-LOX, 5-lypoxigenase; AMPK, AMP kinase; DCF-DA, 2�,7�-dichlorofluorescein diacetate; DPI, diphenyleneiodonium chloride; EGF, epidermalgrowth factor; EGF-r, EGF receptor; gAd, globular adiponectin; Ins-r, insulinreceptor; MAPK, mitogen-activated protein kinase; NAC, N-acetyl cysteine;NADPH, reduced nicotinamide-adenine dinucleotide phosphate; NDGA, nordi-hydroguaiaretic acid; PTP, protein-tyrosine phosphatase; ROS, reactive oxygen spe-cies; RTK, tyrosine kinase receptor.

From the Department of Biochemical Sciences, University of Florence, Florence,Italy.

Received September 27, 2006; accepted January 17, 2007.Supported by the Tuscany Region Studies on Rosiglitazone (TRESOR).Supported by the Italian Association for Cancer Research, the Ministero della

Universita e Ricerca Scientifica e Tecnologica (MIUR-PRIN 2004), ConsorzioInteruniversitario Biotecnologie, and Cassa di Risparmio di Firenze.

Address reprint requests to: Paola Chiarugi, Dipartimento di Scienze Biochim-iche, viale Morgagni 50, 50134 Firenze, Italy. E-mail: paola.chiarugi@unifi.it;fax: (39) 055-4598905.

Copyright © 2007 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.21643Potential conflict of interest: Nothing to report.Supplementary material for this article can be found on the HEPATOLOGY website

(http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

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phosphorylation of acetyl coenzyme-A carboxylase, glu-cose uptake, and lactate production in myocytes.8 In theliver, adiponectin causes a reduction in molecules in-volved in gluconeogenesis accompanied with an increaseof both glycogen synthesis and aerobic glucose consump-tion.9,10 Finally, the adaptor protein APPL1 (AdaptorProtein containing Pleckstrin homology domain, Phos-photyrosine binding domain and Leucine zipper motif)has been recently identified as the first signaling moleculethat binds to the adiponectin receptors and positively me-diates adiponectin signaling in muscle cells.11 Althoughall these metabolic activities induced by adiponectinmight account for the hepatic contribution to the increasein insulin sensitivity in vivo and reduction of in vivo glu-cose levels, their specific signal transduction regulation isstill largely unknown.

Past studies demonstrate that hydrogen peroxide ac-tively participates in several biological processes, includ-ing cell growth, induction, and maintenance of thetransformed state, programmed cell death, and cellularsenescence.12 Several studies revealed that a redox-depen-dent signaling is engaged by several extracellular stimuli,including epidermal growth factor (EGF),13 platelet-de-rived growth factor,14,15 insulin,16 vascular endothelialgrowth factor,17 and integrin receptors.18 Hydrogen per-oxide is a mild oxidant and may oxidize cysteine residuesin proteins to cysteine sulfenic acid or disulfide, both ofwhich are readily re-reduced to cysteine by various cellularreductants. Hydrogen peroxide-susceptible proteins in-clude several transcription factors, such as the p21Rasfamily of proto-oncogenes, which are activated throughoxidation and protein tyrosine phosphatases (PTPs) thatare conversely inactivated by oxidation.19,20 The func-tional relevance of reactive oxygen species (ROS)-medi-ated PTP inhibition in growth factor signaling has beendemonstrated by blocking their accumulation using manyantioxidant drugs.21,22 Redox signaling, therefore, hasbeen correlated with the transient negative regulation ofPTPs, which represents a strategy adopted by cells to pro-mote receptor tyrosine kinase (RTK) signaling by simplyavoiding its prompt inactivation by PTPs.19 Such revers-ible oxidation has been reported for the activation ofmany RTKs by different PTPs, as well as PTP1B forinsulin receptor (Ins-r)23,24 and EGF receptor (EGF-r)13

and LMW-PTP (Low Molecular Weight-PTP) andSHP2 (src homology 2 phosphatase) for platelet-derivedgrowth factor receptor.25,26

We report that in hepatic cells, adiponectin signaling islinked to a transient generation of ROS, which is mainlydriven by activation of 5-lypoxigenase (5-LOX). This ox-idant burst leads to a ligand-independent trans-activationof Ins-r through the oxidation/inactivation of the tyrosine

phosphatase PTP1B and mediates both the intracellularsignaling and the metabolic effects induced by gAd inliver.

Materials and Methods

Materials. Hepa1-6 cells were a gift from ProfessorAndrea Galli (Firenze, Italy); HepG2 and C2C12 cellswere obtained from the American Type Culture Collec-tion (Manassas, VA). The plasmids codifying the mutatedform of Rac1 were a generous gift from Professor Chris-tophe Deroanne (Nice, France). Unless specified, all re-agents were obtained from Sigma, except 2�,7�-dichlorofluorescein diacetate (DCF-DA) (MolecularProbes); polyvinylidene difluoride membrane (Milli-pore); anti-Ins-r, anti-phospho-Y1162-Y1163 of Ins-ranti-PTP1B, anti-AMPK antibodies (Santa Cruz Bio-technology); anti-phosphotyrosine 4G10 antibodies (Up-state Biotechnology); anti–mitogen-activated proteinkinase (MAPK); and anti-phospho-MAPK, anti-phos-pho-p38, anti-p38, and anti–phospho-AMPK antibodies(Cell Signaling Technology Inc.). Globular and full-length adiponectins were obtained from Alexis. Recom-binant PTP1B (50 kDa) was a gift from Dr. Paolo Paoli(Firenze, Italy).

Cell Culture and Transfection. Cells were culturedin Dulbecco’s modified Eagle medium supplementedwith 10% fetal bovine serum in 5% CO2-humidified at-mosphere. For transient transfections, pBABE-GFP-wt,pBABE-GFP-RacV12, and pBABE-GFP-RacN17 plas-mids expressing green fluorescent protein fused with Rac1were purified using a QIAfilter Plasmid Midi kit (Qiagen)and transfected using GeneJammer Transfection Reagent(Stratagene) according to the manufacturer’s instructions.

Immunoprecipitation and Western Blot Analysis.1 � 106 cells were lysed for 20 minutes on ice in 500 �l ofcomplete RIPA lysis buffer (0.1% SDS, 0.5% deoxy-cholate, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1%Nonidet P-40, 2 mM EGTA, 1 mM sodium orthovana-date, 1 mM phenyl-methanesulfonyl-fluoride, 10 �g/mlaprotinin, 10 �g/ml leupeptin). Lysates were clarified viacentrifugation and immunoprecipitated for 4 hours at4°C with 1-2 �g of the specific antibodies. Immune com-plexes were collected on protein A sepharose, separatedvia SDS-PAGE, and transferred onto a polyvinylidenedifluoride membrane. Immunoblots were obtained as de-scribed27 and analyzed with a Biorad ChemiDoc-It Imag-ing System for dedicated chemiluminescent imageacquisition.

Intracellular H2O2 Assay. Hepa1-6 and HepG2 cellswere serum-deprived for 24 hours and then stimulatedwith insulin or gAd, and 3 minutes before the end of

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stimulation, 5 �M DCF-DA was added. Cells were lysedin 1 ml of RIPA buffer (without SDS and deoxycholate)containing 1% Triton X-100 and fluorescence was ana-lyzed immediately using a Perkin Elmer fluorescencespectrophotometer (excitation wavelength, 488 nm;emission wavelength, 510 nm). The values of fluorescencewere normalized on the protein content. For generationof ROS in vivo, Hepa1-6 cells were serum-deprived for 24hours and, where indicated, cells were stimulated withgAd (1 �g/ml) for 15 minutes in the dark. DCF-DA wasadded to the cells 3 minutes before the end of stimulation.Images of ROS-induced DCF fluorescence were capturedusing a confocal fluorescence microscope (Leica TCSSP5).

Determination of Rac1 Activity. After stimulation,Hepa1-6 cells were directly lysed in RIPA buffer and thenclarified via centrifugation. Rac1-GTP was quantified inprecleared protein lysates. Briefly, lysates were incubatedwith 10 �g of PAK-GST fusion protein absorbed on glu-tathione-sepharose beads for 3 hours at 4°C. Immunore-active Rac1 linked to PAK-GST which was thenquantified via anti-Rac1 western blot analysis.

In-Gel Phosphatase Assay. For detection of PTP ac-tivity, we prepared a 10% SDS-polyacrylamide gel con-taining 105 cpm/ml of [32P]-labeled substrate as describedpreviously.28 In the modified version of the assay, whichhas been reported to detect oxidized PTPs,29 10 mM io-doacetic acid was added to the samples after degassing thebuffer. Immunodepleted samples had been subjected to aprevious immunoprecipitation with specific antibodies,and the clarified lysate was analyzed via in-gel assay. Afterelectrophoresis, gels were sequentially washed for the in-dicated times at room temperature with the followingbuffers: buffer 1 (overnight), 50 mM Tris (pH 8.0) and20% isopropanol; buffer 2 (twice, for 30 minutes), 50mM Tris (pH 8.0) and 0.3% �-mercaptoethanol; buffer3 (90 minutes), 50 mM Tris (pH 8.0), 0.3% �-mercap-toethanol, 6 M guanidine hydrochloride, and 1 mMEDTA; buffer 4 (3 washes for 1 hour each), 50 mM Tris(pH 8.0), 0.3% �-mercaptoethanol, 1 mM EDTA, and0.04% Tween 20; buffer 5 (overnight), 50 mM Tris (pH8.0), 0.3% �-mercaptoethanol, 1 mM EDTA, 0.04%Tween 20, and 4 mM dithiothreitol. Gels were thenstained with Coomassie brillant blue, destained in 40%methanol and 10% acetic acid, dried, and analyzed usinga Cyclone system.

Glucose Oxidation. Glucose oxidation was measuredby the production of 14CO2 from D-[U-14C]glucose asdescribed,30 with few modifications. Briefly, cells werecultured in 6-well dishes, serum-deprived for 24 hours,and then incubated with the medium containing 0.2�Ci/ml D-[U-14C]glucose. Each dish had a taped piece of

Whatman paper facing the inside of the dish. The What-man paper was wetted with 100 �l of phenylethylamine-methanol (1:1) to trap the CO2 produced during theincubation period. After the incubation, 200 �l of 4 MH2SO4 was added to the cells, which were then incubatedfor 1 hour at 37°C. Finally, the pieces of Whatman paperwere removed and transferred to scintillation vials for ra-dioactivity counting.

Glycogen Synthesis. Cells were grown until theyreached 60%-80% confluence and were then serum-de-prived for 24 hours. Cells were rinsed in serum-free mediaplus 1% bovine serum albumin and then incubated in thepresence of [3H]-D-glucose (0.4 �Ci/ml) with or withoutthe gAd. Cells were washed with cold phosphate-bufferedsaline and lysed in 1 ml of 2 M NaOH. Exogenous carrierglycogen (10 mg) was added to the sample, and totalglycogen was precipitated with 66% ethanol overnight at�20°C. The glycogen precipitate was recovered via cen-trifugation, resuspended in 200 �l of water, and trans-ferred to scintillation vials for radioactivity counting. Thevalues were then normalized on protein content.

Results

gAd Stimulation Generates a Transient Burst ofROS in Hepatic Cells. Although the metabolic effects ofgAd on liver are well known, the intracellular signalingpathways elicited by this hormone are poorly understood.To investigate the possibility that gAd, as well as manyother growth factors and hormones,19,31 could actthrough redox regulation of intracellular targets, we firststudied hydrogen peroxide production using the mem-brane-permeable fluorescent dye DCF-DA. gAd treat-ment of murine hepatic Hepa1-6 cells leads to a transientincrease in ROS generation, showing a maximum at 15minutes after hormone administration (Fig. 1A). Thecomparison of ROS released upon treatment with insulin,which has been acknowledged to signal through theseoxidant molecules,16 reveals that the 2 hormones elicit asimilar extent of oxidants, although insulin-induced pro-duction is more rapid (Fig. 1B). In addition, we observeda synergistic effect of gAd on insulin-stimulated ROS pro-duction (Matteo Parri, personal communication). Con-versely, full-length adiponectin is less effective in elicitingROS production (data not shown). Data have been con-firmed in human HepG2 cells (Fig. 1C). To characterizethe source of intracellular production of ROS followinggAd stimulation, we analyzed the effect of selective inhib-itors of several enzymes endowed with acknowledged pro-duction of reactive oxygen species (ROS). We useddiphenyleneiodonium chloride (DPI) for reduced nicoti-namide-adenine dinucleotide phosphate (NADPH) oxi-

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dase,32 nordihydroguaiaretic acid (NDGA) or AA861 for5-LOX,33 and rotenone for mitochondrial electron trans-fer chain (complex I). Both NDGA and AA-861 are ableto almost completely block the production of ROS in-duced by gAd, whereas DPI and rotenone are virtuallyineffective (Fig. 1D). Real-time confocal microscopeanalysis further confirms the efficacy of the sole 5-LOXinhibitor NDGA on oxidant production elicited by gAd,thus confirming 5-LOX as the main contributor for ROSincrease in response to the hormone (Fig. 1E).

The small GTPase Rac1 has been involved in the reg-ulation of both 5-LOX and NADPH oxidase activity as adownstream signaling target of several membrane recep-tors, including G-protein–coupled receptors, RTKs, and

integrin receptors.14,18,34 Consistent with these findings,we observed that in liver cells, gAd leads to a strong acti-vation of the small GTPase Rac1, in strict concomitancewith the rise of ROS (Fig. 1F). Furthermore, in keepingwith the current literature describing Rac1 activation andthe subsequent ROS production as a downstream eventwith respect to the activation of phosphatidylinositol3-kinase (PI3K),35 we observed that the phosphatidylino-sitol 3-kinase inhibitor LY294002 is able to abolish bothRac1 activation and ROS production upon gA stimula-tion (Supplementary Fig. 1).

To further investigate the involvement of the smallGTPase Rac1 during gAd signaling, we analyzed the pro-duction of ROS in Hepa1-6 cells transiently overexpress-

Fig. 1. gAd stimulation generates a transient burst of ROS in hepatic cells. (A) ROS production in murine Hepa1-6 cells upon gAd stimulation.Cells were serum-deprived for 24 hours and then stimulated for the indicated periods with gAd (1 �g/ml). *P � 0.001 versus control. **P � 0.005versus control. (B) ROS generation in murine Hepa1-6 cells after insulin stimulation. Cells were treated as described in panel A except for stimulationwith insulin (10 nM). *P � 0.001 versus control. (C) ROS production in human HepG2 cells upon stimulation with human gAd (1 �g/ml). Theexperiment was performed as in panel A. *P � 0.001 versus control. (D) Identification of the source of gAd-driven ROS. Cells were treated as inpanel A. Before adding the hormone, cells were pretreated for 20 minutes with the specific inhibitors of intracellular ROS sources (5 �M DPI, 10�M NDGA, 15 �M AA-861, 5 �M rotenone). *P � 0.001 versus control. (E) Real-time confocal analysis of Hepa1-6 cells upon gAd stimulation.Serum-starved cells were stimulated with gAd in the dark for 15 minutes and DCF-DA were added 3 minutes before the time of stimulation. Imagesof ROS-induced DCF fluorescence were captured by using a confocal fluorescence microscope. (F) gAd-driven oxidant release requires Rac1activation. Hepa1-6 cells were serum-deprived for 24 hours and then stimulated with gAd (1 �g/ml) for the indicated periods. Stimulated cells werethen lysed and processed as described in Materials and Methods. Active Rac1 (top) and total Rac1 (bottom) were quantified via anti-Rac1 westernblotting. The bar graph illustrates the ratio of active Rac1 versus total Rac1 obtained by the mean of 3 independent experiments. *P � 0.001 versuscontrol. (G) Modulation of ROS generated by gAd stimulation through Rac1. Hepatic cells were transfected with RacV12 or RacN17 plasmids.Forty-eight hours after transfection, cells were serum-deprived for 24 hours, and the production of hydrogen peroxide was assayed using DCF-DA asdescribed in (A). The lower panel illustrates the expression of transfected Rac1. *P � 0.001 gAd versus control and V12 � gAd versus V12. *P �0.005 V12 versus control. All data are representative of at least 3 independent experiments.

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ing the constitutively active (RacV12) and the dominantnegative (RacN17) Rac1 mutants. The results reveal thatRac1 functional inhibition through its dominant negativeoverexpression leads to the abolishment of the ROS raiseowing to gAd, while the constitutively active GTPase ec-topic expression leads to an enhancement of ROS, affect-ing both their basal and stimulated levels (Fig. 1G).Together, these findings strongly confirm that the smallGTPase Rac1, in addition to other hormones signalingthrough H2O2, is an upstream regulator involved in thegAd-driven ROS generation.

gAd-Induced Redox Signaling Leads to PTP1B Re-dox Regulation. Because PTPs are intracellular targets ofredox signaling, we analyzed the redox state of PTPs dur-ing gAd stimulation of hepatic cells. We performed amodified PTP in-gel assay that permits, using iodoaceticacid, irreversible alkylation of cysteine residues within theactive site of the in vivo reduced PTPs.29 On the contrary,oxidized PTPs are not alkylated. An aliquot of lysates wassubjected to SDS-PAGE in a gel containing a radioac-tively labeled substrate followed by the renaturation of theproteins in the presence of a reducing agent. Under theseconditions, the activity of nonalkylated/oxidized PTPswas recovered and visualized by the appearance of a clear,white area of dephosphorylation, nearby the position ofthe PTP in the gel. We observed that 2 PTPs of an appar-ent molecular weight of �50 and �60 kDa undergo in-cremental oxidation upon both insulin and gAdtreatment (Fig. 2). On the basis of the molecular weightand the reported redox regulation of PTP1B during insu-lin signaling,23,24 we hypothesize a redox regulation ofPTP1B during gAd treatment as well. We therefore im-munodepleted PTP1B from previously alkylated gAd-treated cell lysates, then performed a modified PTP in-gelassay. PTP1B immunodepletion causes a strong decrease

of both �50- and �60-kDa phosphatases, suggestingthat both PTPs are reactive toward anti-PTP1B antibod-ies, while only the �50-kDa PTP runs as purified recom-binant PTP1B. The identity of PTP1B has beenconfirmed by immunoprecipitating PTP1B in gAd- andinsulin-stimulated samples and modified in-gel PTP assay(Fig. 2B). The presence of 2 different bands of PTP1Bcould be addressed to different oxidation states of thephosphatase. Considering that PTP1B has already beenfound in multiple active fragments in in-gel assays,29

likely due to different posttranslational modifications, wefinally support the implication of PTP1B redox regula-tion during gAd signaling.

gAd Leads to a Redox-Dependent, Ligand-Indepen-dent Tyrosine Phosphorylation of Ins-r. Signal trans-duction integration as been reported to be helped bycross-talk between receptors, frequently leading to RTKtrans-phosphorylation.36 Given the known ability ofPTP1B to regulate Ins-r tyrosine phosphorylation, to-gether with the effect of adiponectin as an insulin-sensi-tizing hormone, we investigated whether gAd can lead totrans-phosphorylation of Ins-r in liver cells. Our resultsindicate that gAd is actually able to cause a ligand-inde-pendent phosphorylation of Ins-r (Fig. 3A). Converselyfull-length adiponectin is not able to induce this phenom-enon (Supplementary Fig. 2). Ins-r activation reaches amaximum within 15 minutes and declines thereafter,demonstrating a transient trans-activation, in keepingwith the increase in ROS level. Again, we confirmed thefinding on human HepG2 cells, which show an earliertime course (Fig. 3B). To further analyze the ligand-inde-pendent trans-phosphorylation of Ins-r induced by gAd,we used phosphospecific antibodies against Tyr1162 andTyr1163 of Ins-r, recently indicated as the main targets ofPTP1B.37 The results indicate a specific ligand-indepen-

Fig. 2. gAd stimulation leads to oxidation of PTP1B. Serum-starved Hepa1-6 cells were exposed to gAd (1 �g/ml) or insulin (10 nM) for theindicated times. (A) Lysates were prepared under anaerobic conditions in the presence of 10 mM iodoacetic acid and then subjected to modifiedin-gel PTP assay. Immunodepletion (Id) of PTP1B has been performed on a set of total lysates after iodoacetic acid treatment. Thirty nanograms ofrecombinant PTP1B was run as a control. The data are representative of three independent experiments. (B) Hepa1-6 cells were treated as describedin panel A, and anti-PTP1B immunoprecipitates (Santa Cruz Biotechnology) were then analyzed via modified in-gel PTP assay. All data arerepresentative of at least three independent experiments.

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dent trans-phosphorylation of these 2 residues upon gAdstimulation of liver cells, thus supporting the possible in-volvement of PTP1B (Fig. 3C). In particular, the detec-tion of phosphorylation of these 2 residues 5 minutes aftergAd stimulation may indicate an earlier effect of PTP1Bwith respect to that observed with total anti-phosphoty-rosine immunoblot. To investigate whether adiponectin-elicited ROS increase is functionally involved in Ins-rtrans-activation, we selectively inhibited the adiponectin-engaged oxidant supply, 5-LOX, using NDGA and AA-861. The results indicate that both inhibitors stronglyinterfere with adiponectin-induced Ins-r phosphorylationon Tyr1162/Tyr1163, thus suggesting a key role ofPTP1B redox regulation in this event (Fig. 3D).

gAd Stimulation Leads to Redox Inhibition of Ins-r–Recruited PTP1B. To identify the PTPs involved inligand-independent Ins-r trans-activation, we performeda nonmodified (i.e., nonredox) in-gel assay analysis onanti–Ins-r immunoprecipitates upon gAd and insulinstimulation. We observed that gAd induces the associa-tion to Ins-r of a pattern of at least four PTPs, similar tothe ligand-dependent one. Of note, one of these Ins-r–associated PTPs comigrates with purified recombinant

PTP1B (Fig. 4A). Furthermore, to detect the redox stateof these PTPs, we performed again a modified (i.e., redoxanalysis) in-gel assay, directly on Ins-r immunoprecipi-tates (Fig. 4B). The results confirm that upon gAd treat-ment, as well as upon insulin stimulation, only the PTP of�50 kDa, comigrating with purified recombinantPTP1B, is transiently oxidized. The identity of PTP1B asthe Ins-r–associating PTP upon gAd treatment has beenconfirmed by immunoblot analysis of Ins-r immunopre-cipitates (Fig. 4C), thus validating our hypothesis of aredox regulation of Ins-r–associated PTP1B during gAdsignaling of liver cells.

ROS Produced in Response to gAd Are EssentialMediators of Its Downstream Effects. To investigatethe role played by ROS in adiponectin function in liver,we first focused on p42 and p44 MAPK as the maintargets of the hormone in Hepa1-6 cells. Indeed, in keep-ing with other reports,7,8,38,39 we observed that inHepa1-6 cells, gAd fails to activate AMPK andp38MAPKs, whereas it is able to elicit the activation ofthese 2 pathways in C2C12 muscle cells (SupplementaryFig. 3). We treated hepatic cells with DPI to inhibit theirNADPH oxidase and NDGA to target their 5-LOX, or

Fig. 3. gAd stimulation trans-phosphorylates Ins-r in hepatic cells. (A) Hepa1-6 cells were serum-starved for 24 hours and then stimulated withgAd (1 �g/ml) or insulin (10 nM) for the indicated periods. Tyrosine phosphorylation level of Ins-r was assayed on Ins-r immunoprecipitates usinganti-phosphotyrosine antibodies (4G10; Upstate Biotechnology). (B) The same experiment described in (A) was repeated in human hepatic HepG2cells, which were stimulated with human gAd (1 �g/ml) or insulin (10 nM) for the indicated periods. (C) Hepa1-6 cells were serum-starved for 24hours and then stimulated with gAd (1 �g/ml) or insulin (10 nM) for the indicated periods. Total lysates (30 �g) were immunoblotted and treatedwith anti-phospho–Ins-r (pY1162/pY1163) antibodies (Santa Cruz Biotechnology) and anti–Ins-r antibodies (Santa Cruz Biotechnology) fornormalization. (D) Cells were treated as in panel C. Before stimulation, cells were pretreated for 20 minutes with NDGA (10 �M) or AA-861 (15 �M).Total lysates (30 �g) were immunoblotted with anti-phospho–Ins-r (pY1162/pY1163) antibodies and anti–Ins-r antibodies for normalization. The dataare representative of three independent experiments, one of which is shown. *P � 0.001 versus control. n.r., nonrelated band.

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with the general ROS scavenger N-acetyl cysteine (NAC).The anti–phospho-MAPK immunoblot revealed thatboth NAC and NDGA cause a decrease of gAd-inducedMAPK activation, whereas the signaling elicited by insu-lin is sensitive to NAC and DPI (Fig. 5A). These findingssuggest a key role of redox signaling in both gAd andinsulin MAPK activation, although it is likely that the 2hormones engage different enzymatic suppliers of oxi-dants (i.e., 5-LOX for gAd and NADPH oxidase for in-sulin). Although the involvement of 5-LOX in

adiponectin signaling is an original data, the activation ofNADPH oxidase in insulin signaling has already beenreported.40

It is reported that adiponectin administration decreasesplasma glucose levels by suppressing hepatic glucose produc-tion.1,10 In addition, in muscle gAd stimulates glucose con-sumption through increased glycogen synthesis and aerobicglycolysis rate.41 We report that in hyperglycemic conditions(medium containing 4500 mg/l glucose), gAd is also able toelicit the storage of glucose into glycogen (Fig. 5B) and theconversion of glucose into CO2 through aerobic oxidation(Fig. 5C) in the liver. To address the role of ROS as intracel-lular regulators of glucose homeostasis in liver cells stimu-lated with gAd, we investigated the outcome of treatmentwith 5-LOX inhibitors. Both the synthesis of glycogen andthe conversion of glucose into CO2 are greatly sensitive toboth NDGA and AA861, thus confirming the engagementof 5-LOX in the signaling pathways elicited by gAd to driveglucose use by liver.

DiscussionWe describe a novel signaling pathway triggered by gAd

in liver cells involving (1) a transient burst of ROS throughthe activation of 5-LOX, (2) the oxidation/inactivation ofPTP1B, (3) a redox-dependent trans-phosphorylation ofIns-r, and (4) a redox-based regulation of downstream sig-naling and metabolic effects of the hormone.

Several growth factors, cytokines, and extracellular ma-trix proteins have been reported to signal through redoxsignaling.19,20 We now include gAd among these extracel-lular stimuli. Redox signaling has been described as en-gaged by a great variety of receptors, including G protein–coupled receptors, RTKs, and integrins, and appears tonot be restricted to a particular type of receptors. Theinclusion of gAd receptors (which do not belong to any ofthe above-mentioned subclasses) among redox-signalingreceptors is thus not surprising and suggests that ROS arewidely used intracellular messengers. The 2 forms of adi-ponectin—full-length and globular—are recognized byAdipoR1 and AdipoR2 receptors. We observed that inliver cells only gAd is able to elicit a great synthesis ofoxidants, whereas the full-length hormone is less effective.The tissue distribution of AdipoR2 shows a strict restric-tion to liver, while AdipoR1 is widely distributed and ispredominant in skeletal muscle. In addition, althoughboth globular and full-length adiponectins bind to bothreceptors, gAd shows an higher affinity for AdipoR1 he-patic receptors.7 The specificity of gAd for redox signalingmay be explained by the ability of the 2 forms of thehormone, likely through binding to different receptorsubsets, to elicit different signals.

Fig. 4. gAd leads to the transient oxidation of PTP1B and to itsrecruitment by Ins-r. (A) Hepa1-6 cells were serum-starved for 24 hoursand then stimulated with gAd (1 �g/ml) or with insulin (10 nM) for theindicated periods. Anti–Ins-r immunoprecipitates were run on radioactiveSDS gels and subjected to a nonmodified (nonredox) in-gel assay todetect the recruitment of PTPs to Ins-r. Immunoprecipitation control withnonrelated IgG is shown. Thirty nanograms of recombinant PTP1B wasrun as a control. (B) Serum-starved Hepa1-6 cells were exposed to gAd(1 �g/ml) or to insulin (10 nM) for the indicated times. Anti–Ins-r immu-noprecipitates were prepared under anaerobic conditions in the presence of10 mM iodoacetic acid and then subjected to a modified PTP in-gel assayto detect the redox state of the phosphatases. Thirty nanograms of recom-binant PTP1B was run as a control. (C) Hepa1-6 cells were stimulated as in(A), and anti-PTP1B and anti–Ins-r immunoprecipitation was performed.These experiments were repeated at least 3 times.

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In keeping with all other reports describing the ability ofgrowth factors and cytokines to transiently affect the regula-tion of intracellular targets through oxidation, gAd redoxsignaling greatly influences the function of the hormone inliver. The intracellular targets of ROS include several pro-teins with reactive cysteine residues and a low pKa that arevulnerable to oxidation by hydrogen peroxide.31 Among re-dox susceptible proteins, PTPs represent enzymes whose ac-tivity can be easily modulated by endogenous ROS. PTPscontain in the catalytic site a cysteine residue that can beoxidized by various oxidants, leading to reversible enzymaticinactivation. Such regulation has been demonstrated forPTP1B during EGF and insulin stimulation22,23 and lowmolecular weight PTP and Src-homology-2 domain upontreatment with platelet-derived growth factor.25,26 Further-

more, a redox regulation of lipid phosphatase PTEN wasrecently reported upon peptide growth factor stimulation.42

We identify PTP1B as the only PTP vulnerable to gAd oxi-dative burst. The redox regulation of the phosphatase doesnot differ for extent and time course between gAd and insu-lin, in keeping with the known similarity in many effectselicited by the 2 hormones.

Many studies have illustrated that the production of ROSis important for best tyrosine phosphorylation and signalingin response to various stimuli. Generation of ROS, throughredox regulation of PTPs, facilitates a tyrosine phosphoryla-tion–dependent cellular signaling response by transiently in-activating those PTPs that normally suppress the signal.19

PTP1Band TC45 (the 45-kDa spliced variant of the T cellprotein-tyrosine phosphatase) have been reported to un-dergo a reversible redox regulation upon insulin signaling.24

The oxidation/inhibition of these phosphatases, recognizingthe �-subunit of the insulin receptor as a substrate, causes thetransient hyperphosphorylation of Ins-r, thus grantingdownstream insulin signaling propagation.24,37 The mostconvincing data about the involvement of PTP1B in insulinsignaling emerge from studies in 2 independently derivedlines of transgenic mice, showing that PTP1B is a key regu-lator of insulin action in vivo. Mice lacking PTP1B exhibitenhanced insulin sensitivity due to increased Ins-r phosphor-ylation in both liver and muscle.43,44 Moreover, the suppres-sion by antisense oligonucleotides of PTP1B expression inanimals model of insulin resistance enhances insulin sensitiv-ity and normalizes blood glucose levels.45-47 Our data showevidence that gAd leads to a redox-dependent trans-phos-phorylation of Ins-r, owing to transient oxidation of Ins-rrecruited PTP1B. In response to gAd, at least four PTPsassociate with Ins-r, although only PTP1B undergoes tran-

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Fig. 5. gAd-driven ROS are essential for MAPK activation, glycogen

synthesis, and CO2 production in hepatic cells. (A) Hepa1-6 cells wereserum-starved for 24 hours and then pretreated with inhibitors of intra-cellular ROS sources (5 �M DPI, 10 �M NDGA) or 20 mM NAC, ageneral scavenger of ROS. Cells were then stimulated with gAd (1 �g/ml)or insulin (10 nM) for the indicated periods. Total lysates (30 �g) wereimmnunoblotted and treated with anti–phospho-42/p44-MAPK antibod-ies (Cell Signalling) and anti–p42/p44-MAPK antibodies (Cell SignalingTechnology) for normalization. The bar graph shows the mean of the ratioof phosphorylated p42/p44-MAPK versus total p42/p44-MAPK obtainedin three independent experiments. *P � 0.001 versus untreated cells.**P � 0.01 versus untreated cells. (B) Hepa1-6 cells were serum-deprived for 24 hours and then stimulated with gAd (1 �g/ml). Whereindicated, AA-861 was added to the cells 20 minutes before stimulation.Glycogen incorporation was assayed as described in Materials andMethods. The values are presented as the percent increase with respectto control and are the mean of 3 independent experiments. *P � 0.001versus gAd 2 hours and gAd 3 hours. **P � 0.05 versus gAd 4 hours.(C) Hepa1-6 cells were treated as described in (B), and CO2 productionat 2 hours was performed as reported in Materials and Methods. Thevalues are presented as the percent increase with respect to control andare the mean of three independent experiments. *P � 0.001.

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sient oxidation, thus suggesting a specificity in its redox reg-ulation and pointing to this phosphatase for ligand-independent Ins-r activation. Remarkably, the ligand-dependent (i.e., upon insulin stimulation) and the ligand-independent (i.e., upon gAd stimulation) Ins-r activationcause the association of a similar set of PTPs, but their oxi-dation appears to be slightly different. Meng et al.24 reportedthe redox regulation of both PTP1B and TC45 during insu-lin ligand-dependent signaling. In our analysis, we con-firmed the appearance of an extra band below PTP1B, likelyTC45 phosphatase, only in insulin-treated samples. Thissuggests a differential involvement of redox regulation ofPTPs during ligand-dependent and -independent Ins-r sig-naling. Conversely, it is likely that ligand-dependent and-independent Ins-r signaling share the tyrosine residues to bephosphorylated. We show evidence that trans-activation in-duced by gAd causes the phosphorylation of 2 tyrosines inposition 1162-1163, which have been indicated as the maintargets of PTP1B during insulin signaling.37

The data presented herein suggest that, besides their func-tion as downstream modulators of RTK signaling upon li-gand binding, ROS act as upstream key molecules in RTKtrans-activation, leading to a ligand-independent signaltransduction. RTK ligand-independent trans-activation hasbeen reported for many RTKs, although the involvement ofredox signaling has not always been implicated. The involve-ment of ROS has been proposed as a causal event for intra-cellular G-protein–coupled receptor–mediated RTK trans-activation, even if the redox regulation of not only PTPs butalso PTKs has been causally implicated (reviewed by Chia-rugi and Giannoni20). In addition, RTKs can be activated intrans by integrins or other neighboring RTKs during theirsignaling. For instance, cross-communication between theMet receptor tyrosine kinase or integrins and EGF-r haveboth been proposed to involve redox signaling.48,49 Finally,hydrogen peroxide has been implicated in lateral propaga-tion of EGF signaling waves, another example of ligand-independent activation of EGF-r phosphorylation.50 In allthese reports, the redox inhibition of PTPs is coupled withEGF-r activity, leading to ligand-independent trans-activa-tion of neighboring EGF-r molecules. With this perspective,we now include Ins-r among RTKs undergoing ligand-inde-pendent trans-activation in response to the stimulation ofneighboring gAd receptors.

Finally, we showed that ROS are key mediators of down-stream metabolic effects elicited by gAd in the liver. In ourcellular model, MAPK activation by gAd is decreased bytreatment with both the general scavenger NAC and with5-LOX inhibitors, suggesting an involvement of gAd-drivenROS in the activation of this pathway in hepatic cells. In theliver, the effect of adiponectin in decreasing glucose bloodlevel has been correlated with its ability to decrease glucone-

ogenesis and to increase glucose oxidation and glycogen syn-thesis. In parallel, adiponectin is able to increase glucoseuptake in muscle. All these effects are likely concurring incontrolling glucose blood levels and have been indicated askey events of adiponectin antidiabetic effects.1,9,10 Data de-rived from mouse models indicate that the decreased level ofadiponectin in lipoatrophic and obese mice correlates withthe development of insulin resistance. In these conditions,the treatment of the animals with gAd determines an in-creased fatty acid combustion and a great increase of Ins-rphosphorylation in muscle.6 Our data indicate a role of gAdredox signaling in hepatic glucose use in response to hyper-glycemic conditions (i.e., increasing both the synthesis ofglycogen and CO2 production). In this view, our results con-tribute to a new explanation of the role of gAd in liver. It isindeed likely that, in addition to the effect elicited in muscleof increased energy expenditure, an insulin-mimetic actionof gAd in the liver would play a role in hyperglicemic condi-tions. It is therefore likely that ROS play a pivotal role in gAdsignaling, being involved in PTP1B-mediated ligand-inde-pendent Ins-r trans-phosphorylation and in the control ofglucose metabolism.

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