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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.
35
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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.
Pathway Coupling Drives Selectivity of IR Agonist 37
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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.
Pathway Coupling Drives Selectivity of IR Agonist 39
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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.
40 Bedinger et al.
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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.
Pathway Coupling Drives Selectivity of IR Agonist 41
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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.
References
Bhaskar V, Goldfine ID, Bedinger DH, Lau A, Kuan HF, Gross LM,
Handa M,Maddux BA, Watson SR, Zhu S, et al. (2012) A fully human,
allosteric monoclonalantibody that activates the insulin receptor
and improves glycemic control. Di-abetes 61:1263–1271.
Bhaskar V, Lau A, Goldfine ID, Narasimha AJ, Gross LM, Wong S,
Cheung B, WhiteML, and Corbin JA (2013) XMetA, an allosteric
monoclonal antibody to the insulinreceptor, improves glycaemic
control in mice with diet-induced obesity. DiabetesObes Metab
15:272–275.
Ceresa BP, Kao AW, Santeler SR, and Pessin JE (1998) Inhibition
of clathrin-mediated endocytosis selectively attenuates specific
insulin receptor signal trans-duction pathways. Mol Cell Biol
18:3862–3870.
Ceresa BP and Pessin JE (1998) Insulin regulation of the Ras
activation/inactivationcycle. Mol Cell Biochem 182:23–29.
Currie CJ, Poole CD, and Gale EA (2009) The influence of
glucose-lowering therapieson cancer risk in type 2 diabetes.
Diabetologia 52:1766–1777.
Cusi K, Maezono K, Osman A, Pendergrass M, Patti ME,
Pratipanawatr T, DeFronzoRA, Kahn CR, and Mandarino LJ (2000)
Insulin resistance differentially affects thePI 3-kinase- and MAP
kinase-mediated signaling in human muscle. J Clin
Invest105:311–320.
Defronzo RA (2009) Banting Lecture. From the triumvirate to the
ominous octet:a new paradigm for the treatment of type 2 diabetes
mellitus. Diabetes 58:773–795.
De Meyts P (2008) The insulin receptor: a prototype for dimeric,
allosteric membranereceptors? Trends Biochem Sci 33:376–384.
Dong XC, Copps KD, Guo S, Li Y, Kollipara R, DePinho RA, and
White MF (2008)Inactivation of hepatic Foxo1 by insulin signaling
is required for adaptive nutrienthomeostasis and endocrine growth
regulation. Cell Metab 8:65–76.
Ellis L, Clauser E, Morgan DO, Edery M, Roth RA, and Rutter WJ
(1986) Re-placement of insulin receptor tyrosine residues 1162 and
1163 compromisesinsulin-stimulated kinase activity and uptake of
2-deoxyglucose. Cell 45:721–732.
Fiory F, Alberobello AT, Miele C, Oriente F, Esposito I, Corbo
V, Ruvo M, Tizzano B,Rasmussen TE, Gammeltoft S, et al. (2005)
Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by
the insulin receptor is necessary for insulin metabolic
signaling.Mol Cell Biol 25:10803–10814.
Frasca F, Pandini G, Scalia P, Sciacca L, Mineo R, Costantino A,
Goldfine ID, BelfioreA, and Vigneri R (1999) Insulin receptor
isoform A, a newly recognized, high-affinity insulin-like growth
factor II receptor in fetal and cancer cells. Mol Cell
Biol19:3278–3288.
Garg SK, Hirsch IB, and Skyler JS (2009) Insulin glargine and
cancer—an un-substantiated allegation. Diabetes Technol Ther
11:473–476.
Glendorf T, Stidsen CE, Norrman M, Nishimura E, Sørensen AR, and
Kjeldsen T(2011) Engineering of insulin receptor isoform-selective
insulin analogues. PLoSONE 6:e20288.
Grouven U, Hemkens LG, Bender R, and Sawicki PT (2010) Risk of
malignancies inpatients with diabetes treated with human insulin or
insulin analogues. Reply toNagel JM, Mansmann U, Wegscheider K, et
al. [letter] and Simon D [letter]. (Letter)Diabetologia
53:209–211.
Hansen BF, Danielsen GM, Drejer K, Sørensen AR, Wiberg FC, Klein
HH,and Lundemose AG (1996) Sustained signalling from the insulin
receptor afterstimulation with insulin analogues exhibiting
increased mitogenic potency. Bio-chem J 315:271–279.
Hansen BF, Kurtzhals P, Jensen AB, Dejgaard A, and Russell-Jones
D (2011) InsulinX10 revisited: a super-mitogenic insulin analogue.
Diabetologia 54:2226–2231.
Hemkens LG, Grouven U, Bender R, Günster C, Gutschmidt S, Selke
GW,and Sawicki PT (2009) Risk of malignancies in patients with
diabetes treatedwith human insulin or insulin analogues: a cohort
study. Diabetologia 52:1732–1744.
Hilgenfeld R, Seipke G, Berchtold H, and Owens DR (2014) The
evolution of insulinglargine and its continuing contribution to
diabetes care. Drugs 74:911–927.
Hubbard SR (2013) The insulin receptor: both a prototypical and
atypical receptortyrosine kinase. Cold Spring Harb Perspect Biol
5:a008946.
Ish-Shalom D, Christoffersen CT, Vorwerk P, Sacerdoti-Sierra N,
Shymko RM, NaorD, and De Meyts P (1997) Mitogenic properties of
insulin and insulin analoguesmediated by the insulin receptor.
Diabetologia 40 (Suppl 2):S25–S31.
Issafras H, Bedinger DH, Corbin JA, Goldfine ID, Bhaskar V,
White ML, Rubin P,and Scannon PJ (2014) Selective allosteric
antibodies to the insulin receptor for thetreatment of
hyperglycemic and hypoglycemic disorders. J Diabetes Sci Technol
8:865–873.
Janssen JA and Varewijck AJ (2014) Insulin analogs and cancer: a
note of caution.Front Endocrinol (Lausanne) 5:79.
Jensen M, Hansen B, De Meyts P, Schäffer L, and Ursø B (2007)
Activation of theinsulin receptor by insulin and a synthetic
peptide leads to divergent metabolic andmitogenic signaling and
responses. J Biol Chem 282:35179–35186.
Jiang ZY, Lin YW, Clemont A, Feener EP, Hein KD, Igarashi M,
Yamauchi T,White MF, and King GL (1999) Characterization of
selective resistance to in-sulin signaling in the vasculature of
obese Zucker (fa/fa) rats. J Clin Invest104:447–457.
Johnson GL and Lapadat R (2002) Mitogen-activated protein kinase
pathways me-diated by ERK, JNK, and p38 protein kinases. Science
298:1911–1912.
Johnson JA and Yasui Y (2010) Glucose-lowering therapies and
cancer risk: the trialsand tribulations of trials and observations.
Diabetologia 53:1823–1826.
Kahn CR (1978) Insulin resistance, insulin insensitivity, and
insulin unresponsiveness:a necessary distinction. Metabolism 27
(Suppl 2):1893–1902.
Kahn CR (1985) The molecular mechanism of insulin action. Annu
Rev Med 36:429–451.
42 Bedinger et al.
at ASPE
T Journals on January 22, 2020
jpet.aspetjournals.orgD
ownloaded from
http://jpet.aspetjournals.org/
-
Kenakin T, Watson C, Muniz-Medina V, Christopoulos A, and Novick
S (2012) Asimple method for quantifying functional selectivity and
agonist bias. ACS ChemNeurosci 3:193–203.
Knudsen L, De Meyts P, and Kiselyov VV (2011) Insight into the
molecular basis forthe kinetic differences between the two insulin
receptor isoforms. Biochem J 440:397–403.
Kurtzhals P, Schäffer L, Sørensen A, Kristensen C, Jonassen I,
Schmid C, and Trüb T(2000) Correlations of receptor binding and
metabolic and mitogenic potencies ofinsulin analogs designed for
clinical use. Diabetes 49:999–1005.
Maegawa H, McClain DA, Freidenberg G, Olefsky JM, Napier M,
Lipari T, Dull TJ,Lee J, and Ullrich A (1988) Properties of a human
insulin receptor with a COOH-terminal truncation. II. Truncated
receptors have normal kinase activity but aredefective in signaling
metabolic effects. J Biol Chem 263:8912–8917.
Malaguarnera R, Sacco A, Voci C, Pandini G, Vigneri R, and
Belfiore A (2012) Pro-insulin binds with high affinity the insulin
receptor isoform A and predominantlyactivates the mitogenic
pathway. Endocrinology 153:2152–2163.
McClain DA, Maegawa H, Levy J, Huecksteadt T, Dull TJ, Lee J,
Ullrich A,and Olefsky JM (1988) Properties of a human insulin
receptor with a COOH-terminal truncation. I. Insulin binding,
autophosphorylation, and endocytosis.J Biol Chem 263:8904–8911.
Mîinea CP, Sano H, Kane S, Sano E, Fukuda M, Peränen J, Lane WS,
and LienhardGE (2005) AS160, the Akt substrate regulating GLUT4
translocation, has a func-tional Rab GTPase-activating protein
domain. Biochem J 391:87–93.
Moller DE, Yokota A, Caro JF, and Flier JS (1989)
Tissue-specific expression of twoalternatively spliced insulin
receptor mRNAs in man. Mol Endocrinol 3:1263–1269.
Morcavallo A, Genua M, Palummo A, Kletvikova E, Jiracek J,
Brzozowski AM, IozzoRV, Belfiore A, and Morrione A (2012) Insulin
and insulin-like growth factor IIdifferentially regulate endocytic
sorting and stability of insulin receptor isoform A.J Biol Chem
287:11422–11436.
Mosthaf L, Grako K, Dull TJ, Coussens L, Ullrich A, and McClain
DA (1990) Func-tionally distinct insulin receptors generated by
tissue-specific alternative splicing.EMBO J 9:2409–2413.
Nagel JM, Mansmann U, Wegscheider K, and Röhmel J (2010) Insulin
resistance andincreased risk for malignant neoplasms: confounding
of the data on insulin glar-gine. Diabetologia 53:206–208.
Nicolucci A (2010) Epidemiological aspects of neoplasms in
diabetes. Acta Diabetol47:87–95.
Pollock RF, Erny-Albrecht KM, Kalsekar A, Bruhn D, and Valentine
WJ (2011) Long-acting insulin analogs: a review of “real-world”
effectiveness in patients with type 2diabetes. Curr Diabetes Rev
7:61–74.
Rathanaswami P, Babcook J, and Gallo M (2008) High-affinity
binding measure-ments of antibodies to cell-surface-expressed
antigens. Anal Biochem 373:52–60.
Reaven GM (1988) Banting lecture 1988. Role of insulin
resistance in human disease.Diabetes 37:1595–1607.
Roth RA and Cassell DJ (1983) Insulin receptor: evidence that it
is a protein kinase.Science 219:299–301.
Schwimmer LJ, Huang B, Giang H, Cotter RL, Chemla-Vogel DS, Dy
FV, Tam EM,Zhang F, Toy P, Bohmann DJ, et al. (2013) Discovery of
diverse and functionalantibodies from large human repertoire
antibody libraries. J Immunol Methods391:60–71.
Sciacca L, Cassarino MF, Genua M, Pandini G, Le Moli R,
Squatrito S, and Vigneri R(2010) Insulin analogues differently
activate insulin receptor isoforms and post-receptor signalling.
Diabetologia 53:1743–1753.
Sesti G, Tullio AN, D’Alfonso R, Napolitano ML, Marini MA,
Borboni P, Longhi R,Albonici L, Fusco A, Aglianò AM, et al. (1994)
Tissue-specific expression of twoalternatively spliced isoforms of
the human insulin receptor protein. Acta Diabetol31:59–65.
Shia MA and Pilch PF (1983) The beta subunit of the insulin
receptor is an insulin-activated protein kinase. Biochemistry
22:717–721.
Takata Y, Webster NJ, and Olefsky JM (1991) Mutation of the two
carboxyl-terminaltyrosines results in an insulin receptor with
normal metabolic signaling but en-hanced mitogenic signaling
properties. J Biol Chem 266:9135–9139.
Tan SX, Ng Y, Meoli CC, Kumar A, Khoo PS, Fazakerley DJ,
Junutula JR, Vali S,James DE, and Stöckli J (2012) Amplification
and demultiplexing in insulin-regulated Akt protein kinase pathway
in adipocytes. J Biol Chem 287:6128–6138.
Taniguchi CM, Emanuelli B, and Kahn CR (2006) Critical nodes in
signallingpathways: insights into insulin action. Nat Rev Mol Cell
Biol 7:85–96.
Ussar S, Vienberg SG, and Kahn CR (2011) Receptor antibodies as
novel therapeuticsfor diabetes. Sci Transl Med 3:113ps147.
Vigneri P, Frasca F, Sciacca L, Pandini G, and Vigneri R (2009)
Diabetes and cancer.Endocr Relat Cancer 16:1103–1123.
Vigneri R, Squatrito S, and Frittitta L (2012) Selective insulin
receptor modulators(SIRM): a new class of antidiabetes drugs?
Diabetes 61:984–985.
White MF, Livingston JN, Backer JM, Lauris V, Dull TJ, Ullrich
A, and Kahn CR(1988) Mutation of the insulin receptor at tyrosine
960 inhibits signal transmissionbut does not affect its tyrosine
kinase activity. Cell 54:641–649.
Whitten AE, Smith BJ, Menting JG, Margetts MB, McKern NM,
Lovrecz GO, AdamsTE, Richards K, Bentley JD, Trewhella J, et al.
(2009) Solution structure of ecto-domains of the insulin receptor
family: the ectodomain of the type 1 insulin-likegrowth factor
receptor displays asymmetry of ligand binding accompanied bylimited
conformational change. J Mol Biol 394:878–892.
Wilden PA, Backer JM, Kahn CR, Cahill DA, Schroeder GJ, and
White MF (1990)The insulin receptor with phenylalanine replacing
tyrosine-1146 provides evidencefor separate signals regulating
cellular metabolism and growth. Proc Natl Acad SciUSA
87:3358–3362.
Xie L, Mark Jones R, Glass TR, Navoa R, Wang Y, and Grace MJ
(2005) Measure-ment of the functional affinity constant of a
monoclonal antibody for cell surfacereceptors using kinetic
exclusion fluorescence immunoassay. J Immunol Methods304:1–14.
Yamaguchi Y, Flier JS, Benecke H, Ransil BJ, and Moller DE
(1993) Ligand-bindingproperties of the two isoforms of the human
insulin receptor. Endocrinology 132:1132–1138.
Yamaguchi Y, Flier JS, Yokota A, Benecke H, Backer JM, and
Moller DE (1991)Functional properties of two naturally occurring
isoforms of the human insulinreceptor in Chinese hamster ovary
cells. Endocrinology 129:2058–2066.
Zhao J, Linden E, Der K, Cao L, Shimizu R, Hansen BC, Rubin P,
and Bezwada P(2014) XMetA, a novel insulin receptor activator, is
efficacious in glycemic controlin rhesus monkeys with
naturally-occurring type 2 diabetes, in Proceedings of theAmerican
Diabetes Association 74th Scientific Sessions; 2014 June 13–17;
SanFrancisco, CA. Vol. 63 (Suppl 1) Abstract 123-OR, American
Diabetes Association,Alexandria, VA.
Address correspondence to: Sean H. Adams, Arkansas Children’s
NutritionCenter, 15 Children’s Way, Little Rock, AR 72202. E-mail:
[email protected]
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