differential pathway coupling of the activated insulin...

1521-0103/353/1/35–43$25.00 http://dx.doi.org/10.1124/jpet.114.221309THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 353:35–43, April 2015U.S. Government work not protected by U.S. copyright

Differential Pathway Coupling of the Activated Insulin ReceptorDrives Signaling Selectivity by XMetA, an Allosteric PartialAgonist Antibody

Daniel H. Bedinger, Ira D. Goldfine, John A. Corbin, Marina K. Roell, and Sean H. AdamsXOMA Corporation, Berkeley, California (D.H.B., I.D.G., J.A.C., M.K.R.); Obesity & Metabolism Research Unit, United StatesDepartment of Agriculture–Agricultural Research Service Western Human Nutrition Research Center and Department ofNutrition, Davis, California (S.H.A.); and Molecular, Cellular and Integrative Physiology Graduate Group, University of California atDavis, Davis, California (D.H.B., S.H.A.)

Received November 7, 2014; accepted January 22, 2015

ABSTRACTThe monoclonal antibody XMetA is an allosteric partial agonistof the insulin receptor (IR), which activates the metabolic Aktkinase signaling pathway while having little or no effect on themitogenic extracellular signal‐regulated kinase (ERK) signalingpathway. To investigate the nature of this selective signaling, wehave conducted a detailed investigation of XMetA to evaluatespecific phosphorylation and activation of IR, Akt, and ERK inChinese hamster ovary cell lines expressing either the short orlong isoform of the human IR. Insulin activated both pathways,but the phosphorylation of Akt was more sensitive to the hormonethan the phosphorylation of ERK.Maximally effective concentrations

of XMetA elicited phosphorylation patterns similar to 40–100 pMinsulin, which were sufficient for robust Akt phosphorylation, buthad little effect on ERK phosphorylation. These data indicate thatthe preferential signaling of XMetA is due to an innate difference inpathway sensitivity of Akt versus ERK responses to IR activationand partial agonism by XMetA, rather than a separate pathway-biasedmechanism. Themetabolic selectivity of partial IR agonistslike XMetA, if recapitulated in vivo, may be a desirable featureof therapeutic agents designed to regulate blood glucose levelswhile minimizing undesirable outcomes of excessive IR mitogenicactivation.

IntroductionMost patients with type 2 diabetes mellitus (T2DM) are both

hyperglycemic and resistant to both endogenous and exogenousinsulin (Reaven, 1988; Defronzo, 2009). In many patients, fastinghyperglycemia can be corrected only by providing exogenousinsulin, often in the form of long-acting insulin or a long-acting insulin analog (Pollock et al., 2011; Hilgenfeld et al.,2014). Insulin treatment, while effective, has potential risks,including weight gain, episodic hypoglycemia, and activation ofthe mitogenic insulin signaling pathway (Hemkens et al., 2009;Vigneri et al., 2009; Janssen and Varewijck, 2014). There isalso a proposed link between long-acting insulin therapy and

an increased risk of cancer, but this link is controversial (Currieet al., 2009; Garg et al., 2009; Grouven et al., 2010; Johnson andYasui, 2010; Nagel et al., 2010; Nicolucci, 2010). However,there is relatively strong in vitro evidence that insulin andinsulin analogs increase the growth of tumor cells (Hansenet al., 1996; Kurtzhals et al., 2000; Sciacca et al., 2010). Thesepotential risks of insulin highlight challenges in the manage-ment of T2DM patients with insulin, and the need for newagents that maximize the beneficial metabolic effects of insulinwhile minimizing the negative effects of the hormone.When insulin binds to the insulin receptor (IR), it triggers

a conformational change that allows for the autophosphorylationof tyrosines on the receptor’s intracellular b-subunit (Roth andCassell, 1983; Shia andPilch, 1983; Kahn, 1985; DeMeyts, 2008).These phosphotyrosines serve kinase regulatory functions andbecome binding substrates for SH2 domain–containing adapterproteins, such as the IR substrate (IRS) proteins, Shc, and GRB2(Taniguchi et al., 2006). The canonical metabolic pathway ac-tivation by insulin involves IR autophosphorylation and IRSprotein binding and phosphorylation, followed by the associationand activation of phosphatidylinositol-4,5-bisphosphate 3-kinase,which leads to the activation of phosphoinositide-dependentkinase 1. Then, phosphoinositide-dependent kinase 1 activatesAkt by phosphorylating it at Thr308 (Taniguchi et al., 2006; Tanet al., 2012). Akt kinase is a key enzyme in metabolic insulin

This work was funded by a Cooperative Research and Development Agree-ment [CRADA 58-3K95-1-1497] between XOMA and United States Departmentof Agriculture–Agricultural Research Service (USDA-ARS), as well as througha USDA-ARS Intramural Project [5306-51530-019-00D]. USDA is an equalopportunity provider and employer. D.H.B., I.D.G., J.A.C., and M.K.R. areemployees of XOMA (US), LLC. No other potential conflicts of interest relevantto this article are reported. S.H.A. has no conflict of interest to declare.

Portions of this work were previously presented in a poster at the followingconference: Bedinger D, Corbin J, Roell M, Goldfine I, and Adams S (2014)“Metabolic” vs. “mitogenic” insulin receptor signaling by an allosteric mono-clonal antibody: specific metabolic pathway bias or partial agonism? AmericanDiabetes Association 74th Scientific Sessions; 2014 Jun 13–17; San Francisco,CA.

dx.doi.org/10.1124/jpet.114.221309.

ABBREVIATIONS: BSA, bovine serum albumin; CHO, Chinese hamster ovary; ERK, extracellular signal‐regulated kinase; IR, insulin receptor; IRS,insulin receptor substrate; KinExA, kinetic exclusion assay; T2DM, type 2 diabetes mellitus.

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action (Miinea et al., 2005; Dong et al., 2008). This insulin-mediated signaling cascade acts in various cell types toorchestrate a shift from the use of stored energy in the body(glycogen, fat, and amino acids) to the storage and utilizationof abundant prandial glucose and amino acids.In addition to its metabolic effects, insulin can also trigger the

activation of mitogenic pathways (Vigneri et al., 2009). Phos-phorylated IR and IRS-1, bound by the Shc protein, serve aseffective adaptors for the GRB2-SOS complex, thus activatingRAS and the mitogen-activated protein kinase cascade (Hansenet al., 1996; Ceresa and Pessin, 1998). The mitogen-activatedprotein kinase, extracellular signal-regulated kinase (ERK1/2;also referred to as p44/p42-activated protein kinase), is a keyregulator of the mitogenic response to insulin (Johnson andLapadat, 2002).IR monoclonal antibodies may represent a novel class of long-

acting therapeutics for regulating glucose metabolism in T2DM(Ussar et al., 2011). Monoclonal antibodies to the IR also havethe potential to elicit metabolic effects while minimizing mi-togenic responses. We have recently reported the developmentof XMetA, a fully humanmonoclonal antibody to the IR (Bhaskaret al., 2012). XMetA is an allosteric partial agonist of the IRthat does not influence either insulin’s binding to the receptor’sorthosteric site or insulin’s ability to activate downstreamsignaling. In cell culture, XMetA stimulates key metabolicfunctions of insulin signaling, including glucose transport.XMetA is also active on the mouse and monkey IR. In rodentmodels of diabetes (Bhaskar et al., 2012, 2013), and inspontaneously diabetic primates (Zhao et al., 2014), XMetAdecreases fasting blood glucose levels. Moreover, XMetA didnot cause hypoglycemia in these diabetic animals (Bhaskaret al., 2012, 2013; Issafras et al., 2014).Unlike insulin, which can stimulate both metabolic and

mitogenic pathways via Akt andERK, respectively, we observedthat XMetA stimulated the Akt metabolic pathway, but gen-erated little or no activation of the mitogenic ERK pathway.Moreover, unlike insulin, XMetA did not induce proliferationof tumor cells (Bhaskar et al., 2012, 2013). This observationsuggested that XMetA activated downstream signaling path-ways of the IR in a manner that differed from that of insulin.Therefore, to fully interpret this selective signaling, in thepresent study a more detailed analysis of the activation prop-erties of XMetA on these different pathways (in comparison withinsulin) has been undertaken. First, studies were performedto determine whether the effects of XMetA on the insulin sig-naling cascade were IR isoform specific. In humans and othermammals, the IR exists in two splice variant isoforms (Frascaet al., 1999). A long form (IR-B) contains the 12 amino acid exon11 segment of the extracellular a-chain and is the dominantisoform in adult insulin target tissues, specifically, liver and fat(Moller et al., 1989; Mosthaf et al., 1990; Glendorf et al., 2011).The shorter isoform, IR-A, which does not contain exon 11,is the predominant isoform in fetal tissues, lymphocytes,endothelium, and many tumor cells, and is also expressedto varying degrees in insulin-sensitive tissues (Moller et al.,1989; Mosthaf et al., 1990; Sesti et al., 1994). Second, tounderstand XMetA signaling via the IR, a more detailedanalysis of XMetA and insulin binding and their activation ofAkt and ERK has been carried out. Our results provideimportant insights into the relative activation of the meta-bolic and mitogenic pathways downstream from the IR by

insulin and XMetA, and may have implications for develop-ment of potential new agents for the treatment of diabetes.

Materials and MethodsEstablishment of Cell Lines. The Chinese hamster ovary (CHO)-K1

cells employed in the current studies contained less than 5000 hamsterIRs and less than 5000 IGF-1Rs (Bhaskar et al., 2012). These CHO-K1cells were transfected with a stable plasmid containing a neomycin-selective marker and either the long form (IR-B) or the short form (IR-A)of the human IR cDNA (Yamaguchi et al., 1991; Bhaskar et al.,2012). The CHO-hIR-A and CHO-hIR-B cells were cloned by limitingdilution, screened by flow cytometry for high expression, and cul-tured in EX-CELL-302 media (Sigma-Aldrich, St. Louis, MO). BothIR-transfected cell lines had approximately 250,000 receptor dimersper cell. The 3T3R-IR-A cells were obtained from the University ofCalifornia at San Francisco (San Francisco, CA) and carry a deletionof the IGF-1 receptor and express approximately 400,000 IR-A receptordimers per cell.

XMetA Binding Assessed by Kinetic Exclusion Assay. Tomeasure the effect of insulin on the binding affinity of XMetA for bothisoforms of the human IR, we employed equilibrium assays underconditions in which there was either no insulin present or a saturatinginsulin concentration (175 nM) present. XMetA (50 pM) was incubatedwith increasing concentrations of CHO cells (maximum 4 � 107cells/ml)for 18 hours on a rotator at 5°C in phosphate-buffered saline with 0.25%bovine serum albumin (BSA) (Sigma-Aldrich) and 0.1% sodium azide(Sigma-Aldrich). Cells were pelleted by centrifugation and the amount offree XMetA remaining in the supernatant solution was measured byimmunofluorescence using a kinetic exclusion assay (KinExA) instru-ment (S�apidyne Instruments, Boise, ID). Antibody concentration datawere fit using KinExA software (Xie et al., 2005; Rathanaswami et al.,2008) (standard affinity curve fit model) to yield an estimate of theequilibrium dissociation (KD) values. Preliminary data indicated thatXMetA binds divalently to the IR dimer, establishing a 1:1 stoichiometrybetween the IgG molecule and receptor dimer (data not shown).

Insulin Binding Assessed by KinExA. To measure the effect ofXMetA on the binding affinity of insulin for both isoforms of the humanIR, we employed equilibrium assays under conditions in which there waseither no XMetA present or where a saturating XMetA concentration(33 nM) was present. Then, 50 pM human insulin (Sigma-Aldrich) andeither XMetA or an anti-keyhole limpet hemocyanin IgG2 isotype controlantibody (33 nM) was incubated for 18 hours on a rotator at 5°C inphosphate-buffered saline with 0.25% BSA and 0.1% sodium azide withincreasing concentrations of either CHO-hIR-A or CHO-hIR-B cells. Cellswere pelleted by centrifugation and the amount of free insulin in thesolution was measured by immunofluorescence using a KinExA in-strument. Briefly, polymethylmethacrylate beads were coated with65 mg/ml D6C4 anti-insulin monoclonal Ab (Fitzgerald Industries, Acton,MA), and the captured insulin was detected with 0.15 mg/ml biotin-labeled D3E7 anti-insulin monoclonal antibody (Fitzgerald Industries)mixed with 1 mg/ml streptavidin-phycoerythrin. Insulin binding datawere fit using KinExA software as described previously to determine theinsulin binding affinity (KD) (Xie et al., 2005; Rathanaswami et al., 2008).This methodology, employed to measure insulin affinity, was designed toanalyze the high-affinity insulin binding site and minimize the influenceof the negative cooperativity effect (DeMeyts, 2008;Whitten et al., 2009).

The Effect of XMetA on Insulin Signaling in CulturedCells. For studies evaluating the activation of insulin signaling events,CHO-K1 cells expressing either the human IR-A or human IR-B isoformwere first incubated in Dulbecco’s modified Eagle’s medium (25 mMglucose)with 0.3%BSA for 5hours to reduce background signals, and thenincubated with increasing concentrations of either insulin or XMetA for10 minutes. In preliminary studies, this time was found to yield a robustresponse to insulin. Both longer and/or shorter incubations were usedin the time course assays, as specified in the text. Cells were pelletedby centrifugation at 4°C, the supernatant was decanted, and the cells

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resuspended in coldTris lysis buffer [150mMNaCl, 20mMTrizma-HCl,pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, phosphataseinhibitors 2 and 3 (100 ml/10 ml), and 2 mM PMSF, all from Sigma-Aldrich], and cOmplete Protease Inhibitor tablets (Hoffman-La Roche,Basel, Switzerland). Samples were then analyzed by Western blot usingantibodies recognizing total Akt, Akt phosphorylated at Thr308, totalErk1/2, Erk1/2 phosphorylated at Thr202/Tyr204, Thr185/Tyr187, IRphosphorylated at Tyr1328, and b-actin (Cell Signaling Technology,Danvers, MA). The total IR b-subunit antibody and the antibody to IRphosphorylated at Tyr1162/1163 were from EMD Millipore (Billerica,MA), and the antibody to IR phosphorylated at Tyr972 was fromInvitrogen (Carlsbad, CA). Sampleswere reduced and run on a 10%2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol gel using4-morpholinepropanesulfonic acid–SDS running buffer, and then trans-ferred to polyvinylidene difluoride membranes. Blots were blocked withTris-buffered saline with 0.1% Tween 20 and 5% BSA, and then probedwith primary antibodies at 1:5000 dilution. Detection was performedwith anti-rabbit IgG(H1L)-HRP (Jackson ImmunoResearch, WestGrove, PA), imaged using SuperSignal West Dura ChemiluminescentSubstrate (Pierce/Thermo Fisher, Waltham, MA) on a ChemiDoc MPcharge-coupled device imager (Bio-Rad, Hercules, CA), quantitated inthe Image Lab software (Bio-Rad) and normalized to percentage ofmaximal insulin response (25 nM insulin) for each marker. Data werefit using Prism software (sigmoidal dose response, variable slope4 parameter fit) (GraphPad Software, La Jolla, CA) to generate EC50values. Lysates were analyzed for IRS-1 phosphorylation by plate-based immunoassay using a kit fromMeso Scale Discovery (Rockville,MD). Analysis of signaling in 3T3R-IR-A cells was performed asdescribed previously with the modification that no centrifugation wasrequired because the cells were adherent and the supernatant wasaspirated. Unless stated otherwise, all figures represent results fromat least three independent experiments, each with either duplicate ortriplicate determinations. Values are reported as the mean 6 S.E.M.

Evaluation of Agonist Bias Using the Black-Leff OperationalModel. Signaling response data were evaluated using the Black-Leffoperationalmodel to calculate log(t/KA) values (Kenakin et al., 2012). Thelog(t/KA) values were calculated using the operational model in Prism(GraphPad Software) and the data are presented as 6 S.E.M.

Results

Binding of XMetA to Cells Expressing Either Isoformof the Human IR (CHO-hIR-A or CHO-hIR-B). Studieswere first carried out in cultured CHO-K1 cells expressingeither the hIR-A or hIR-B isoform to determine the bindingaffinity (KD) of XMetA to each isoform (Fig. 1). XMetA bound tothe hIR-A isoform with an affinity of 55 6 16 pM (Fig. 1A) andbound to the hIR-B isoformwith a nearly identical affinity of 50611 pM (Fig. 1B). The affinity of XMetA to both IR isoforms was

independent of the presence of insulin. This analysis indicatedthat the presence of the region encoded by exon 11 of the IR didnot influence XMetA binding.Binding of Insulin to Cells Expressing Different Forms

of the Human IR (CHO-hIR-A and CHO-hIR-B). Studieswere carried out in cultured CHO-K1 cells to determine theaffinity of insulin to both isoforms of the human IR (Fig. 2).Insulin bound to cells expressing the hIR-A isoform with an af-finity of 156 6 14 pM in the presence of control antibody, and2166 100 pM in the presence of XMetA (Fig. 2A). Insulin boundto cells expressing the hIR-B isoform with an affinity of 221 628 pM in the presence of control antibody and 277 6 112 pMin the presence of XMetA (Fig. 2B). The affinity of insulin to bothIR isoformswas independent of XMetA (P5 0.45). In the absenceof XMetA, the affinity of insulin was slightly higher for the hIR-Aform (P 5 0.024) than for the hIR-B form. This slightly higheraffinity for the hIR-A form is in agreement with previouslypublished results using other techniques (Mosthaf et al., 1990;Yamaguchi et al., 1991, 1993; Sciacca et al., 2010; Knudsen et al.,2011).Effect of Insulin and XMetA on Autophosphorylation

of the IR Kinase Regulatory Domain of the Two hIRIsoforms. Autophosphorylation of IR beta subunit tyrosines1150/1151 of the hIR-A isoform and the beta subunit tyrosines1162/1163 of the hIR-B isoform are necessary to allow activationof IR tyrosine kinase activity (Hubbard, 2013). In the hIR-Aisoform, insulin stimulated this phosphorylation half-maximally(EC50) at 486 6 238 pM (Fig. 3A). The effect of insulin on thehIR-B isoform phosphorylation occurred at slightly lower con-centrations, stimulating this function half-maximally at 118 632 pM (Fig. 3B). The maximal effect of XMetA on IR autophos-phorylation of both receptor isoforms was markedly lower thanthat of insulin, achieving only 20–30% of the maximal effect ofinsulin. XMetA stimulated hIR-A isoform phosphorylationhalf-maximally at 445 6 77 pM (Fig. 3A), and hIR-B isoformphosphorylation half-maximally at 1430 6 42 pM (Fig. 3B).Comparison of the Effects of Insulin and XMetA on

Other Tyrosine Phosphorylation Sites on the IR andIRS-1. Because the IR has additional phosphorylation sites, weevaluated whether the lower maximal autophosphorylationby XMetA was unique to the kinase regulator loop tyro-sines (vide supra). Thus, we also evaluated two other tyrosineautophosphorylation sites representing the juxtamembranedomain (Tyr960 for hIR-A and Tyr972 for hIR-B) and theC-terminal region (Tyr1316 for hIR-A and Tyr1328 for hIR-B)(Fig. 3). Insulin stimulated phosphorylation of the juxtamembrane

Fig. 1. Effect of insulin on XMetA binding to CHO-K1cells expressing either human IR-A or IR-B. Varyingconcentrations of CHO-K1 cells in suspension, express-ing either IR-A (A) or IR-B (B), were incubated in thepresence (filled squares) and absence (open circles) of175 nM insulin plus 50 pM XMetA antibody at 4°C for18 hours. After removal of the cells by centrifugation,the free antibody in the supernatant at equilibriumwas analyzed by immunofluorescence-based KinExA,and then analyzed with KinExA software to determinebinding affinity. Data are the mean 6 S.E.M. fromtriplicate experiments.

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tyrosine of the IR-A isoformwith an EC50 of 6906 141 pM andthe IR-B isoformwith anEC50 of 394688 pM. Insulin stimulatedthe phosphorylation of the C-terminal domain with an EC50 of1.556 0.17 nM for the IR-A isoform and 1.546 0.09 nM for theIR-B isoform. Thus,while the IR-B isoformkinase regulatory looptyrosines were more sensitive to insulin-stimulated auto-phosphorylation than the IR-A isoform, the juxtamembraneand C-terminal tyrosines had similar insulin-stimulated doseresponses in both isoforms.In the CHO-hIR-A cells, XMetA induced the autophosphor-

ylation of all these tyrosines to a level of approximately 20%that of insulin with similar dose responses (Fig. 3A). In theCHO-hIR-B cells, XMetA induced autophosphorylation fortyrosines 972 that was also 20% that of insulin, but the tyrosinenear the C terminus (Tyr1328) had a slightly higher level ofactivation to approximately 40% that of insulin (Fig. 3B).XMetA stimulated phosphorylation of the juxtamembranetyrosine of the IR-A isoformwith an EC50 of 8076 360 pM andthe IR-B isoform with an EC50 of 1.96 6 0.46 nM. XMetAstimulated the phosphorylation of the C-terminal domain withan EC50 of 1.32 6 411 nM for the IR-A isoform and 4.52 60.15 nM for the IR-B isoform. Thus, XMetA was a modestlymore potent activator of IR-A autophosphorylation than IR-Bin terms of dose response; however, the maximal levels ofautophosphorylation induced by XMetA were very similar be-tween the two isoforms.

After autophosphorylation, the IR phosphorylates tyrosineson IRS proteins. Thus, the effect of insulin and XMetA wasalso studied on IRS-1 phosphorylation (Fig. 3C). XMetA ata maximal concentration stimulated IRS-1 phosphorylationat a level approximately 20% that of a maximally effectiveconcentration of insulin.Effect of Insulin on Activation of Akt and ERK in

CHO-IR Cells. We next studied the effect of insulin onphosphorylation of Akt at Thr308, the site required for itskinase activation and activation of IR-mediated metabolicsignaling (Taniguchi et al., 2006). In the cells expressing theIR-A isoform (Fig. 4A), 10 minutes of incubation with insulinstimulated Akt phosphorylation half-maximally at 123 636 pM. In the cells expressing the IR-B isoform (Fig. 4A), insulinstimulated Akt phosphorylation half-maximally at 42 6 10 pM(Fig. 4B).We next studied the effect of insulin on the phosphorylation

of ERK1/2 at Thr202/Tyr204 and Thr185/Tyr187, the sitesrequired for its kinase activation and activation of IR-mediatedmitogenic signaling. To stimulate ERK1/2 activation, muchhigher concentrations of insulin (10- to 20-fold) were requiredwhen compared with insulin stimulation of Akt: in cells ex-pressing the IR-A isoform (Fig. 4A) insulin stimulated ERKphosphorylation half-maximally at 14506 274 pM, and in cellsexpressing the IR-B isoform insulin stimulated ERK phos-phorylation half-maximally at 837 6 188 pM (Fig. 4B).

Fig. 2. Effect of XMetA on insulin binding to CHO-K1cells expressing either human IR-A or IR-B. Varyingconcentrations of CHO-K1 cells, expressing IR-A (A) orthe IR-B (B), were incubated in the presence of either33 nMXMetA (filled squares) or control antibody of thesame isotype (open circles) plus 50 pM insulin at 4°Cfor 18 hours. After removal of the cells by centrifuga-tion, the free insulin in the supernatant at equilibriumwas analyzed by immunofluorescence-based KinExA,and then analyzed with KinExA software to determinethe affinity. Data are the mean 6 S.E.M. from trip-licate experiments.

Fig. 3. Effect of insulin and XMetA on IR autophosphorylation in CHO-hIR-A and hIR-B cells. IR autophosphorylation of the juxtamembrane, kinaseregulatory loop, and C-terminal tyrosines was evaluated in the CHO-IR-A cells (A) at Tyr960 (squares), Tyr1150/1151 (circles), and Tyr1316 (triangles)by either insulin (solid symbols and solid curves) or XMetA (open symbols and dashed curves). The analogous tyrosines were also evaluated forautophosphorylation in CHO-IR-B cells (B) at Tyr972 (squares), Tyr1162/1163 (circles), and Tyr1328 (triangles). CHO cells expressing IR-A or IR-Bisoforms were cultured in growth factor–deficient media for 5 hours, stimulated with various concentrations of either XMetA or insulin for 10 minutes at37°C, pelleted by centrifugation at 4°C, and then lysed. Lysates were analyzed by Western blot using specific IR phosphotyrosine antibodies and imagedon a charge-coupled device imager. Densitometry data were normalized to percentage of maximal insulin response for each phosphotyrosine and shownas the mean 6 S.E.M. from triplicate experiments. (C) Lysates were analyzed for IRS-1 phosphorylation using a plate-based immunoassay (Meso ScaleDiscovery). Four stimulations per column are shown from a single experiment, with the mean 6 S.D.

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Effect of XMetA on Activation of Akt and ERK inCHO-IR Cells. Next, we studied the effect of XMetA on thephosphorylation of Akt at Thr308. Maximal phosphorylation ofthis enzyme was approximately 60% that of insulin for both IRisoforms. In cells expressing the hIR-A isoform (Fig. 4C), XMetAstimulated Akt phosphorylation half-maximally at 3566 60 pM,and in cells expressing the hIR-B isoform, XMetA stimulated Aktphosphorylation half-maximally at 9686 42 pM (Fig. 4D). Thus,when Akt is measured, CHO-hIR-B cells are more sensitive toinsulin and less sensitive to XMetA than the CHO-hIR-A cells.The maximal effect of XMetA on the phosphorylation of

ERK1/2 was only 14% that of insulin in cells expressing thehIR-A isoform (Fig. 4C) and less than 4% that of insulin in cellsexpressing the hIR-B isoform (Fig. 4D). XMetA stimulated ERKphosphorylation in cells expressing the hIR-A isoform half-maximally at 1000 6 190 pM and stimulated ERK phosphor-ylation in cells expressing the hIR-B isoform at 14306 270 pM.Effect of Duration of Incubation on IR Autophosphor-

ylation, Akt Phosphorylation, and ERK Phosphorylation.The effect of duration of incubation was studied on the afore-mentioned functions for both insulin and XMetA. Incubationtimes were 2, 5, 10, and 20 minutes (Fig. 5). The concentrationstested at these times were a maximally effective XMetA concen-tration of 100 nM, a maximally effective insulin concentration of25 nM, and a lower insulin concentration of 100 pM. The lowerconcentration of insulin caused a level of pIR and pAkt activationsimilar to that of 100 nMXMetA. This analysis indicated that thedifferences between XMetA and insulin on the aforementionedparameters were not the result of kinetic differences.Comparison of the Effects of Insulin and XMetA on

3T3R-IR Cells. To explore whether the differential in acti-vation of Akt versus ERK by both insulin and XMetAwas specificfor CHO cells, Akt and ERK activation was investigated ina second cell line, mouse fibroblasts expressing hIR-A. In this cell

line, insulin stimulated Akt activation half-maximally at 81 630 pM (Fig. 6A). XMetA stimulated Akt activation to a level ap-proximately 80% that of insulin (Fig. 6B). The one half-maximalXMetA concentration was 1960 6 244 pM.As with CHO cells expressing the human IR, much higher

concentrations of insulin were required to stimulate ERK1/2activation in the 3T3R-IR cells; the half-maximal insulin con-centrationwas 4736 116 pM (Fig. 6A). For XMetA, themaximalpERK1/2 response was approximately 5% that of insulin (Fig. 6B).These studies indicated that the effect of insulin on ERK ac-tivation also required higher concentrations of insulin whencompared with Akt activation in a second cell line, and thatXMetA had much greater effects on Akt activation than on ERKactivation. Thus, the differential effects of XMetA were notspecific to CHO cells.Evaluation of Agonist Bias Using the Black-Leff

Operational Model. Signaling response data were evaluatedusing the Black-Leff operational model to calculate log(t/KA)values. The log(t/KA) value is a transduction coefficient that canbe used as ameasure of ligand potency in terms of dose response(Kenakin et al., 2012). This transduction coefficient incorporatesboth an affinity factor (KA) and an efficacy factor (t) to estimatethe relative potency of an agonist, and can be calculated for eachactivation pathway or function and used to quantify signalingbias of agonists relative to a reference agonist. The log(t/KA)values were calculated for insulin or XMetA stimulation of Aktand ERK1/2 phosphorylation (Fig. 7, A and B). The Δlog(t/KA)values (Fig. 7, C andD) show the difference in log(t/KA) values ofXMetA from insulin for either the Akt or the ERK pathway.The insulin-subtracted Δlog(t/KA) of the two pathways’ stimu-lation by XMetA have overlapping errors and are not signif-icantly different, demonstrating a lack of agonist bias. Tofurther illustrate this relationship, bias plots were created(Fig. 7, E and F). These plots compare the relative activation

Fig. 4. Effect of insulin andXMetA on phospho-Akt andphospho-ERK1/2 in CHO-hIR-A and CHO-hIR-B cells.Phospho-AKT at Thr308 (circles) and phospho-ERK1/2at Thr202/Tyr204 (squares)were evaluated inCHO-IR-A(A and C) and CHO-IR-B (B and D) cells. Phosphoryla-tion of AKT and ERK was evaluated following stimula-tion with either insulin (A and B) or XMetA (C and D).CHO cells expressing IR-A or IR-B isoforms werecultured in growth factor–deficient media for 5 hours,stimulatedwith various concentrations of either XMetAor insulin for 10 minutes at 37°C, pelleted by cen-trifugation at 4°C, and then lysed. Lysates wereanalyzed by Western blot using either antiphosphoAkt or antiphospho ERK1/2 antibodies, and thenimaged on a charge-coupled device imager. Densitometrydata were normalized to percentage of maximal insulinresponse for each phosphotyrosine and shown as themean 6 S.E.M. from triplicate experiments.


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of the Akt and ERK pathways at the same concentration ofagonist. The XMetA response on the bias plot closely followsthat of insulin for both the hIR-A and hIR-B experiments.This demonstrates that the innate differential sensitivities ofthe ERK and AKT pathways to IR stimulation are sufficient todescribe the pathway activation characteristics observed forXMetA, as opposed to XMetA having a unique differentialpotency for AKT relative to ERK compared with insulin.

DiscussionThemonoclonal antibody, XMetA, was isolated from a human

antibody phage display library (Schwimmer et al., 2013), and

has been shown to bind to both the hIR-A and hIR-B isoforms ofthe receptor. XMetA is an allosteric partial agonist that binds toand activates the IR, and activates the Akt, but not the ERKpathway. Therefore, XMetA behaves as a selective IR modula-tor (Vigneri et al., 2012). The current studies investigated indetail the mechanism(s) whereby XMetA selectively activatesthe Akt pathway. We also determined whether these signalingproperties of XMetA are the same for both the A and B isoformsof the human IR.In the case of insulin, a differential dose response for Akt and

ERK activation via the IR has been previously observed inseveral studies (Jensen et al., 2007; Malaguarnera et al., 2012),but the effects of an IR partial agonist, such as XMetA, on this

Fig. 5. Effect of duration of incubation onIR autophosphorylation, Akt phosphory-lation, and ERK1/2 phosphorylation. IRautophosphorylation at pTyr1162/1163(A), phospho-Akt at Thr308 (B) andphospho-ERK1/2 at Thr202/Tyr204 (C)were evaluated in the CHO-IR-B cell atvarious incubation times with either in-sulin or XMetA. Cells were cultured ingrowth factor–deficient media for 5 hours,and then stimulated with either 25 nMinsulin (open square), 100 pM insulin (opentriangle), 100 nM XMetA (closed square), orcontrol media (closed circle) for the indicatedtimes at 37°C. Cells were then pelletedby centrifugation, and lysed. Lysates wereanalyzed by Western blots using specificantibodies and imaged on a charge-coupleddevice imager (D). Densitometry data werenormalized to percentage ofmaximal insulinresponse for each function. Data from twoindependent experiments were analyzed.

Fig. 6. The effect of insulin and XMetA on phospho-Akt and phospho-ERK1/2 in 3T3R-hIR-A cells.Phospho-Akt at Thr308 (circles) and phospho-ERK1/2 at Thr202/Tyr204 (squares) were evaluatedin the 3T3R-IR-A cells. Cells were cultured in mono-layer with growth factor–deficient media for 5 hours,and then stimulated with various concentrationsof either insulin (A) or XMetA (B) for 10 minutesat 37°C. Supernatants were aspirated and the cellswere lysed. Lysates were analyzed by Western blotusing specific detection antibodies and imaged ona charge-coupled device imager. Data were normalizedto percentage of maximal insulin response for eachphosphotyrosine and are shown as the mean6 S.E.M.from triplicate experiments.

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differential dose response have not been explored. Wemeasuredsignaling functions downstream from the IR: the phosphoryla-tion of Akt, a major regulator of the metabolic effects of insulin,and ERK1/2, amajor regulator of themitogenic effects of insulin.Interestingly, the insulin dose response for Akt phosphorylationand activation occurred at significantly lower hormone concen-trations than that of ERK1/2 phosphorylation. This differentialsensitivity of Akt versus ERK1/2 was seen with both IR isoformsand in several cell types. Thus, these data indicate that the Akt

pathway inherently has greater signal amplification (Tan et al.,2012) than the ERK1/2 pathway following IR activation (Kahn,1978; Ish-Shalom et al., 1997). Previously, we compared theeffect of insulin and XMetA on glucose uptake and cell pro-liferation (Bhaskar et al., 2012). Glucose uptake was enhancedat subnanomolar concentrations by both insulin and XMetA.In contrast, cell proliferation was only enhanced by insulin atnanomolar concentrations and XMetA had no effect on thisparameter. These data are compatible with the observations (1)that insulin and XMetA both activate the IR/Akt metabolicpathway; and (2) that insulin at relatively higher concentrationsactivates the IR/ERK mitogenic pathway, whereas XMetAstimulation of this pathway is negligible.XMetA, as a partial IR agonist, exploits this differential

pathway sensitivity in both isoforms of the IR to elicit anapparent metabolic signaling bias. Because XMetA stimu-lates sufficient IR autophosphorylation to activate Akt, theantibody mimics the metabolic effects of low concentrationinsulin. Because more IR activation is required to activate theERK pathway than the Akt pathway, a compound that iscapable of only partial IR activation would have little or noability to activate the less efficiently coupled ERK pathway.Thus, a major reason why XMetA does not activate ERK isthat, as a partial IR agonist, the antibody does not stimulatesufficient IR autophosphorylation to activate the ERK pathway.When the insulin dose-response curves for these variousmarkers of insulin activation, as well as the maximal levelof stimulation by XMetA, are plotted together for comparison(Fig. 8), it can be demonstrated that XMetA, at its maximumeffective dose, behaves much like a physiologic concentrationof insulin (40–100 pM) in CHO-hIR cells. Therefore, thesedata suggest that the observed metabolic selectivity by XMetAis a direct consequence of its partial agonism combined with thenatural differential signaling pathway sensitivities of Aktversus ERK when stimulated by IR activation. This conclusionis supported by the biased agonist analysis (Fig. 7) as appliedfrom the Black-Leff operational model (Kenakin et al., 2012).Another potential mechanism for differential post-IR signal-

ing by XMetA was alternate phosphorylation of b-subunittyrosines. The IR has seven identified tyrosines that areautophosphorylated by the IR kinase domain (Wilden et al.,1990), and these tyrosines are divided into threemain regions: (1)the juxtamembrane region (965 and 972); (2) the kinase reg-ulatory domain (1158, 1162, and 1163); and (3) the C-terminalregion (1328 and 1334) (Hubbard, 2013). There are no reportsof IR tyrosines that specifically regulate mitogenic activation

Fig. 7. Analysis of agonist bias using the Black-Leff operational model. Dose/response data shown in Fig. 4 were analyzed using the Black-Leff operationalmodel to calculate the log(t/KA) transduction coefficient of insulin or XMetAfor Akt phosphorylation (circles) and ERK phosphorylation (squares) for theCHO-IR-A (A) and CHO-IR-B (B) cells. From these the Δlog(t/KA) values forXMetA for both pathways was calculated in reference to insulin in CHO-IR-A(C) and CHO-IR-B (D) cells. Bias plots are shown comparing the percentageof maximal insulin stimulation of AKT and ERK phosphorylation for insulin(red circles) and XMetA (black squares) at each concentration of agonist forCHO-IR-A (E) and CHO-IR-B (F).

Fig. 8. Working model illustrating thatXMetA behaves similarly to a low-concen-tration of insulin. Phospho-IR (triangle),phospho-Akt (circles), and phospho-ERK(squares) insulin dose-response curves pre-sented inFigs. 3 and 4 have been combined.The horizontal dashed lines are the maxi-mal activation levels achieved by XMetA(100 nM) for the respective markers. Thevertical line highlights a single insulin con-centration that would approximate the max-imal XMetA effect across the three markersof insulin activation.


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because all tyrosine deletions decrease themetabolic responseto insulin (Ellis et al., 1986; Maegawa et al., 1988; McClainet al., 1988;White et al., 1988). We observed that for both IR-Aand IR-B the kinase regulatory domain was most sensitive toinsulin, and the C-terminal region was least sensitive to insu-lin. Mutation and deletion studies regarding the C-terminalphosphotyrosines’ contributions to insulin signaling supportthe notion that the C-terminal tyrosines have amoderate role inactivating metabolic insulin signaling by acting as a bindingsubstrate to the Akt-activating PDK-1 enzyme (Fiory et al.,2005), but have little if any effect on mitogenic insulin signaling(Maegawa et al., 1988;McClain et al., 1988; Takata et al., 1991).In the CHO-hIR-A cells, the dose response of all three sites toXMetA was similar; XMetA was able to only partially stimulatereceptor autophosphorylation to a level about 20% that of in-sulin. In the CHO-hIR-B cells, but not the CHO-hIR-A cells, thetyrosine near the C terminus (1328) had a slightly higher levelof activation to about 40% that of insulin. This difference ismodest and would not be sufficient to describe the Akt selec-tivity observed fromXMetAbecause pathway selectivity occurredin a similar manner for both the hIR-A and hIR-B cells. Thus,it is unlikely that XMetA induced pathway specificity via aselective tyrosine phosphorylation profile.When compared with insulin, several studies have reported

that certain insulin analogs enhance activation of the mitogenicpathway. These studies are difficult to interpret in terms of IRsignaling because different mutants and analogs often haveenhanced activities against the IGF-1R receptor, which con-tributes to the mitogenic response (Hansen et al., 2011). Sciaccaet al. (2010) evaluated insulin analogs in cells that had no IGF-1Rexpression and demonstrated that some analogs maintaineda preference for mitogenic pathway activation when comparedwith insulin. The mechanisms of this preference are not clear,but analogswith a slow receptor dissociation rate demonstratedmore mitogenic signaling (Hansen et al., 1996). The differentaffinities and binding properties of the insulin analogs may beable to effect IR trafficking and internalization, which can influ-ence levels of ERK activation (Ceresa et al., 1998; Morcavalloet al., 2012). However, unlike XMetA, these insulin analogs areall orthosteric binders and, importantly, act as full agonists ofthe IR, with maximal levels of receptor activation similar to thatof insulin. While some analogs have enhancedmitogenic activitycompared with insulin, none have been reported to have en-hanced metabolic activity. Thus, XMetA is a unique moleculein predominately stimulating metabolic activity via the IR.A molecule such as XMetA that has enhanced metabolic

relative to mitogenic activity may be clinically useful. It hasbeen proposed that in most insulin-resistant T2DM subjects,the metabolic pathway of the IR is selectively more resistant toinsulin when compared with the mitogenic pathway (Jianget al., 1999; Cusi et al., 2000). Therefore, a molecule that selec-tively stimulates the metabolic pathway might provide clinicalbenefits, while avoiding untoward effects of high tonic or epi-sodic insulin exposure.

Acknowledgments

The authors thank Diane Wilcock and Patrick Scannon for helpfulreviews of and comments on this manuscript.

Authorship Contributions

Participated in research design: Bedinger, Corbin, Roell, Adams.Conducted experiments: Bedinger.Performed data analysis: Bedinger.

Wrote or contributed to the writing of the manuscript: Bedinger,Goldfine, Adams.

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