Evidence that Receptor Aggregation
may Play a Role in Transmembrane
Signaling through the Insulin-Like
Growth Factor-I Receptor
Nobuhiko Ikari, Hiroko Yoshino, Alan C. Moses, and Jeffrey S. Flier
Charles A. Dana Research Institute
and the Harvard-Thorndike Laboratory of Beth Israel Hospital
Department of Medicine
Beth Israel Hospital and Harvard Medical School
Boston, Massachusetts 02215
alR-3 is a mouse monoclonal antibody that binds to
an epitope on the human insulin-like growth factor I
(IGF-I) receptor and inhibits [125I]IGF-I binding to this
receptor on human skin fibroblasts (HSF) and Hep
G2 human hepatoblastoma cells. Unlike the natural
ligand (IGF-I), neither intact alR-3 nor its monovalent
Fab fragment stimulate aminoisobutyric acid (AIB)
uptake in HSF, and both competitively antagonize
IGF-I's ability to produce this effect. However, when
HSF are incubated with alR-3 or its Fab' fragment,
subsequent exposure to anti-mouse immunoglobulin
G (IgG) produces a potent stimulation of AIB uptake.
Anti-Mouse IgG by itself does not effect AIB uptake.
alR-3 also antagonizes IGF-I's ability to stimulate
glycogen synthesis in Hep G2 cells. As with AIB
uptake in HSF, the combination of «IR-3 followed by
anti-mouse IgG stimulates glycogen synthesis in
Hep G2 cells to the same extent as that produced
by IGF-I. The triggering of these two biological effects depends on the concentration of both alR-3
and anti-mouse IgG. These results are consistent
with the possibility that local aggregation or crosslinking of IGF-I receptors plays an important role in
transmembrane signaling by this receptor. (Molecular Endocrinology 2: 831-837, 1988)
cellular responses that range from nutrient transport to
cellular proliferation (2). The IGF-I receptor consists of
two a-subunits and two /3-subunits linked by disulfide
bridges (3). Both classical biochemistry and, more recently, receptor cDNA cloning, indicate the strong structural and sequence homology of this receptor with the
insulin receptor (3-7). As with insulin and the insulin
receptor, binding of IGF-I to the a-subunit of its own
receptor causes the rapid activation of tyrosine kinase
activity intrinsic to the /?-subunit (8, 9). Little is known,
however, about the precise mechanism whereby binding of ligand to the extracellular a-subunit eventuates
in a transmembrane signal that activates the catalytic
function of the 0-subunit and the subsequent cascade
of intracellular events.
It is well known that autoantibodies to the insulin
receptor, such as those that occur in patients with type
B insulin resistance, both block insulin binding and
trigger certain biological effects of insulin (10-12). Using
purified immunoglobulin G (IgG) from serum of one such
patient, it was previously shown that, unlike intact IgG
or Fab2 fragments, monovalent anti-insulin receptor
antibody (Fab') behaved as a competitive antagonist of
insulin action in rat adipocytes. However, the insulinlike activity of monovalent autoantibody could be restored by the addition of a second antibody (13). These
observations led to the initial suggestion that local
aggregation or cross-linking of insulin receptors may
play an important role in the transmembrane signaling
process (13).
Recently, a mouse monoclonal antibody to the human
IGF-I receptor («IR-3) has been produced (14). alR-3
binds to an epitope distinct from that to which IGF-I
itself binds and inhibits the binding of IGF-I but not of
insulin to a variety of human cells (15,16). This antibody
also inhibits several biological responses to IGF-I (1719). Since intact alR-3 lacks intrinsic IGF-I agonist
activity (19), we have evaluated whether receptor
bound alR-3 can be induced to trigger a transmembrane signal by exposing this antireceptor antibody to
anti-mouse immunoglobulin.
INTRODUCTION
Insulin-like growth factor I (IGF-I) is a single chain polypeptide hormone that is structurally homologous to
proinsulin (1). IGF-I binds to a specific membrane receptor (type I IGF receptor) which has high affinity for
IGF-I, and a much lower affinity for insulin (2). Activation
of this receptor in target cells triggers a variety of
0888-8809/88/0831 -0837$02.00/0
Molecular Endocrinology
Copyright © 1988 by The Endocrine Society
831
MOL ENDO-1988
832
Vol 2 No. 9
RESULTS
Effect of Cross-Linking of alR-3 on AIB Uptake
Inhibition of 125I-IGF-I Binding to Two Human Cell
Types by alR-3
Since «IR-3 alone did not stimulate AIB uptake in HSF,
we next evaluated whether anti-mouse IgG, when
added as a second antibody would cross-link «IR-3 and
stimulate AIB uptake. When cells were exposed to
varying concentrations of «IR-3 for 1 h at 37 C, washed,
and then incubated with 5 tiQ/m\ anti-mouse IgG, a
biphasic curve for stimulation of AIB uptake was observed. Stimulation increased to a maximum of 155%
of control at 3 jug/ml of «IR-3 and then declined toward
control levels at higher concentrations of «IR-3 (Fig. 3).
This effect also was dependent on the concentration of
anti-mouse IgG. To determine the optimum concentra-
We first compared the ability of purified alR-3 to inhibit
125
I-IGF-I binding to human skin fibroblasts (HSF) in
suspension and to HEP G2 hepatoblastoma cells in
monolayer. Binding of IGF-I to HSF was studied in cells
suspended from monolayer by gentle trypsinization
since this method markedly reduces the high content
of IGF binding protein that is associated with the surface
of HSF monolayers and that interferes with IGF-I binding
to its receptor (20-22). As anticipated, when «IR-3 was
coincubated with 125I-IGF-I, «IR-3 inhibited IGF-I binding
in both cell types in a dose-dependent manner (Fig. 1).
The ED50 was approximately 60 ng/ml in HSF and 20
ng/ml in Hep G2 cells.
300
Inhibition of IGF-I-Stimulated AIB Uptake by alR-3
in HSF
We previously reported that «IR-3 produced a dosedependent inhibition of IGF-I stimulated aminoisobutyric
acid (AIB) uptake when it was coincubated with IGF-I
and HSF (19). In the current experiments, we have
altered this protocol by first incubating HSF with «IR-3
for 1 h, removing «IR-3 not associated with the cells,
and then exposing the cells to IGF-I. As anticipated, 20
ng/ml IGF-I stimulated [3H]AIB uptake in HSF to 250%
of basal uptake (Fig. 1). «lR-3 at 10 ng/m\ by itself did
not stimulate AIB uptake in HSF. When monolayers of
HSF were incubated with 10 /*g/ml «IR-3 for 1 h at 37
C, washed, and further incubated with 20 ng/ml IGF-I
for 2 H, IGF-I's ability to stimulate AIB uptake was
inhibited by 32% (Fig. 2). This degree of inhibition was
statistically significant but less than that seen when
alR-3 and IGF-I were incubated together rather than
sequentially (19).
100-1 HSF IN SUSPENSION
50
BASAL
IGF-I
IR-3
IGF+IR-3
INCUBATION CONDITIONS
Fig. 2. Effect of «IR-3 on AIB Uptake by HSF Monolayer
Monolayer cultures of HSF were studied according to procedures described in Materials and Methods. AIB uptake is
assessed as percent of basal stimulation. Data is the mean ±
SEM for three separate experiments done in triplicate. Incubation conditions are listed below each bar. Basal, No additives;
IGF-I, 20 ng/ml IGF-I; «IR-3, 10 ^g/ml alR-3; IGF + IR-3, 10
fig/m\ alR-3 plus 20 ng/ml IGF-I.
_
1
HEP G2 CELLS
•D
.E 80£1
ED 50 ~ 60 ng/ml
±. a.
CD v>
o
1 40 H
J5 «
2 E
•s 20-
10 c
10'
10 z
10
ALPHA IR-3 (ng/ml)
10"
50
100
150
ALPHA IR-3 (ng/ml)
Fig. 1. Inhibition of [125I]IGF-I Binding to HSF in Suspension and HEP G2 Cells in Monolayer by «IR-3
Left panel, HSF in suspension. Right panel, HEP G2 cells in monolayer. Data from a representative experiment is normalized to
percent control binding. Each point is the mean of triplicate determinations of percent specific binding in each experiment.
Receptor Aggregation and Transmembrane Signaling
833
180-1
16U-
" p<0.01
" p<0.01
140-
IR-3 +ANTI-MOUSE IgG
T /
L/
/
120-
x
\
\
ANTI-MOUSE IgG + IR-3
160
•
J
5
CD _
< O
[
120
ANTI-MOUSE IgG ALONE
100-
f
—
80-
10' 3
10"2
10"1
>|r
i
100-
IR-3 ALONE
1—
10°
i
10 1
•—•-
1
80
10 z
15
ALPHA IR-3 (ug/ml)
Fig. 3. Effect of Cross-Linked «IR-3 on AIB Uptake.
Monolayer cultures of HSF were preincubated with «IR-3
for 1 h. After being washed, cells were incubated with either
5 Mg/ml anti-mouse IgG (D—D) or no anti-mouse IgG ( • — • )
for 2.5 h at 37 C. AIB uptake is plotted as percent of basal
uptake vs. the log concentration of «IR-3. Data is the mean ±
SEM for three separate experiments done in triplicate. *, Statistically significant difference (P < 0.01) between incubations
done in the presence and absence of anti-mouse IgG.
tions of anti-mouse IgG, cells were preincubated with a
fixed concentration of «IR-3 (10 Mg/ml) for 1 h at 37 C,
washed, and then incubated with increasing amounts
of anti-mouse IgG. As shown in Fig. 4, the combination
of antibodies clearly increased AIB uptake above basal.
Peak stimulation to 165% of basal occurred at 5 Mg/ml
anti-mouse IgG.
To assess whether the valency of «IR-3 determined
its ability to stimulate AIB uptake in the presence of
anti-mouse IgG, we next tested the effect of «IR-3 Fab'
fragments cross-linked by anti-mouse IgG to stimulate
AIB uptake. Fab' inhibited the binding of 125I-IGF-I in
HSF suspension (ED50 = 500 ng/ml), although with a
lower potency than intact «IR-3 (data not shown). Fab'
also inhibited AIB uptake stimulated by 20 ng/ml IGF
by 30% at a concentration of 50 Mg/ml. When cells
were preincubated with increasing concentrations of
Fab' (Fig. 5), washed, and then incubated with 5 Mg/ml
anti-mouse IgG for 2 h at 37 C, a significant increase
of AIB uptake occurred. Neither Fab' alone nor antimouse IgG alone stimulated AIB uptake. The optimum
concentration of Fab' was 5
Inhibition of IGF-I-Stimulated Glycogen Synthesis
by alR-3 in HEP G2 Cells
Verspohl et al. (23) have demonstrated that IGF-I stimulates glycogen synthesis in the human hepatoblastoma cell line HEP G2 in part through the IGF-I receptor.
We have used this model system to investigate whether
the ability of cross-linked «IR-3 to stimulate a biological
response is specific for only one cell and one biological
response or whether it is a more generalized phenomenon. First, we tested the effect of «IR-3 on glycogen
25
35
45
55
ANTI-MOUSE IgG (ug/ml)
Fig. 4. Dose dependence of Anti-Mouse IgG
Monolayer culture of HSF were preincubated with either 10
Mg/ml «IR-3 (•—CI) or no «IR-3 (M-M) for 1 h, followed by
washing and by addition of increasing amounts of anti-mouse
IgG. After 2.5 h incubation, AIB uptake was performed. AIB
uptake is plotted as percent of basal vs. concentration of
antimouse IgG. Data are mean ± SE of two individual experiments. " , Statistical differences (P < 0.01) in the presence
and absence of alR-3.
200
Fig. 5. Effect of alR-3 Fab' Alone and Cross-Linked with AntiMouse (Anti-M) IgG on AIB Uptake.
Monolayer cultures of HSF were incubated with basal medium (column 1), 20 ng/ml IGF-I (column 2), 20 ng/ml IGF-I
plus 50 alR-3 Fab' (column 3), 50 /xg/ml Fab' alone (column
4), 5 Mg/ml rabbit anti-mouse IgG (column 5), or 50 M g «IR-3
Fab' plus 5 Mg/ml anti-mouse IgG (column 6). AIB uptake is
plotted as percent of basal uptake. Data is the mean ± SE of
two individual experiments.
synthesis stimulated by IGF-I. Twenty nanograms of
IGF-I stimulated glycogen synthesis to 185% of basal.
dR-3 alone did not stimulate glycogen synthesis. The
pretreatment of HEP G2 cells with 10 Mg/ml «IR-3 for
1 h substantially inhibited glycogen synthesis stimulated
by 20 ng/ml IGF-I (Fig. 6).
MOL ENDO-1988
834
Vol 2 No. 9
200
•*p<0.01
IR-3 + ANTI-MOUSE IgG
160-
z =_ (a
UJ
Z Si
UJ _
O
IR-3 ALONE
BASAL
IGF-I (20 ng)
IR-3
IGF-I + IR-3
INCUBATION CONDITIONS
Fig. 6. Effect of alR-3 on Glycogen Synthesis by Hep G2 Cells
Monolayer cells of Hep G2 were studied according to the
procedure described in Materials and Methods. Glycogen synthesis is normalized to percent of basal. Each value is the
mean of two individual experiments. The additives for each
incubation are indicated below each bar. Bar 1, Basal; bar 2,
IGF-I at 20 ng/ml; bar 3, «IR-3 at 10 Mg/ml; bar 4, alR-3 at 10
plus IGF-I at 20 ng/ml.
5
15
25
ALPHA IR-3 (ug/ml)
Fig. 7. Effect of Cross-Linked alR-3 on Glycogen Synthesis
by Hep G2 Cells
Hep G2 were preincubated with increasing amounts of «IR3 for 1 h followed by washing with basal medium and addition
of [3H]D-glucose with either 5 /*g/ml anti-mouse IgG (•—•) or
no anti-mouse IgG (•—•) for 2 h. Data are normalized to
percent of basal and given as the mean ± SE of three individual
experiments. *, Statistical differences (P < 0.01) in the presence and absence of anti-mouse IgG. Bar on the right, Glycogen synthesis stimulated by 20 ng/ml IGF-I.
Effect of Cross-Linked <*IR-3 on Glycogen
Synthesis
HEP G2 monolayer cells were incubated with increasing
amounts of «IR-3 for 1 h, washed, and incubated with
5 Mg/ml anti-mouse IgG and D-[U-14C]glucose for 2 h at
37 C. This concentration of anti-mouse IgG plus 10 /xg
alR-3 gave maximal stimulation of AIB transport in HSF.
The combination of the two antibodies stimulated glycogen synthesis to a level equivalent to that produced
by 20 ng/ml IGF-I (Fig. 7).
DISCUSSION
Antireceptor antibodies, whether spontaneously arising
in patients with autoimmune disease or experimentally
derived, have been useful tools in studies of receptor
structure and function. One area in which these antibodies have been useful is in the study of the mechanism of receptor signaling. Antireceptor antibodies and
natural receptor ligands do not always have the same
effects upon cellular function. Receptor antibodies may
function as full or partial agonists, simulating the normal
response to the ligand, as antagonists, or as both,
depending on the duration of exposure.
The factors that determine whether an anti-receptor
antibody will be an agonist or not have yet to be defined,
and this is in part due to our limited understanding of
the molecular mechanism by which the binding of the
natural ligand to an extracellular receptor domain activates a transmembrane signal, such as a change in the
activity of a receptor tyrosine kinase. Some insights
into this question have emerged from earlier studies
with spontaneously occurring anti-insulin receptor antibodies (11-13). The IgG fraction of serum from one
such patient was shown to be a full receptor agonist in
a variety of cell types, and this activity was preserved
in divalent (Fab2) but not monovalent (Fab') antibody
preparations (13). Importantly, the monovalent Fab'
antireceptor antibody, which by itself behaved as a
competitive antagonist of insulin action, regained agonist activity after exposure to anti-IgG (13). This second
antibody was presumed to exert this effect by inducing
receptor aggregation or cross-linking, an event that has
been invoked as participating in the actions of insulin
(24) and several other ligands (25-27).
The experiments presented in the current report using the monoclonal antibody to the type I IGF receptor,
alR-3, differ in some respects from the foregoing observations in the insulin receptor system, but in general
support similar conclusions. Thus, unlike the polyclonal
insulin receptor autoantibodies, which are potent receptor agonists, intact «IR-3 does not exert an agonistic
effect, but behaves instead as a competitive antagonist
in two distinct cell types each measuring a different
biochemical effect. This finding indicates that «IR-3 is
incapable of inducing the transmembrane signal merely
by binding to the type I IGF receptor. Thus, these data
are analogous to prior observations with the Fab' fraction of anti-insulin receptor IgG (13). Likewise, the fact
that addition of anti-IgG converts intact «IR-3 into an
agonist is also parallel to observations with Fab' fragments of insulin receptor antibodies.
We and others previously have shown that alR-3
probably binds to an epitope on the type I IGF receptor
that is not identical to the IGF-I binding site (14-16).
Receptor Aggregation and Transmembrane Signaling
Thus, while alR-3 blocks IGF-I binding and biological
effects, it can precipitate type I IGF receptors to which
125
I-IGF-I has been chemically cross-linked (14). Furthermore, IGF-I is a poor competitor for the binding of
[125l]«IR-3 to the receptor (15). We have interpreted
these results as being consistent with alR-3 inhibiting
IGF-I binding by steric hindrance after binding to a site
close to but not identical to the IGF-I binding site.
Why is it that the Fab' fragment of the insulin receptor
antibody and intact bivalent «IR-3 IgG are devoid of
agonist activity, whereas each can be "activated" by
addition of a second antibody? It is clear in both cases
that simple binding of the antibody to the receptor can
be dissociated from the conformational change that is
presumed to follow hormone binding, ultimately causing
an increase in j8-subunit autophosphorylation and activation of subsequent steps. This presumed conformational change requires a precise fit of receptor and
ligand that the antireceptor antibody binding to its discrete epitope cannot mimic. Are there any mechanistic
implications of the second antibody induced agonistic
effect? The most conservative implication is that the
binding of a second antibody to the receptor bound
antireceptor antibody somehow induces a change in
the conformation of the receptor that brings about the
signaling event. This could involve intra- or intermolecular events. Thus, the critical event could be a conformational change within the receptor induced by binding
of the second antibody. Alternatively, it could be the
case that intermolecular receptor aggregation is the key
conformational event involved in signal transduction
that is brought about by the second antibody. In the
latter case, the failure of intact «IR-3 to act could have
several different explanations. These include: 1) the
antibody binding epitope does not trigger the proper
conformational change that brings about aggregation
or; 2) the physical presence of the antibody blocks the
necessary aggregation of receptors, but this is relieved
by cross-linking molecules of «IR-3 with a second antibody. Our observations do not resolve whether intraor intermolecular events of the level of the receptor
determine these biological effects.
Do these events relate to the ability of antireceptor
antibodies to trigger receptor autophosphorylation? In
preliminary studies using solubilized IGF-I receptors
from HEP G2 cells (Ikari, N., unpublished data), neither
intact alR-3 nor cross-linked antibody were .able to
mimic the action of IGF-I to induce receptor autophosphorylation. These results, if confirmed by additional
studies on solubilized receptors and on intact cells,
would have implications for the role of receptor autophosphorylation in IGF action, but they do not shed any
light on the mechanism by which antibody binding does
or does not transmit a conformational signal to the /3subunit in the interior of the cell.
In summary, the binding of this monoclonal antibody
to IGF-I receptors can provoke IGF-I actions, but unlike
most insulin receptor antibodies, this is only seen when
the receptor-antibody complex is further perturbed by
the binding of a second antibody. It therefore is obvious
that an alteration in the conformation of the IGF-I recep-
835
tor is one mechanism by which this receptor can initiate
a biological response. Further studies will be necessary
to determine if IGF-I activation of the IGF-I receptor
induces conformational changes in the receptor similar
to those induced by cross-linked antireceptor antibody.
MATERIALS AND METHODS
Reagents and hormones were obtained from the following
sources: culture dishes from Costar (Cambridge, MA), Dulbecco's Modified Eagle's Medium from Gibco (Grand Island, NY),
calf serum from M.A. Bioproduct, [3H]AIB and D-[U- 14 C] glucose from New England Nuclear [(NEN), Boston, MA]. Recombinant human Thr^-IGF-I was purchased from Amgen (Thousand Oaks, CA), rabbit liver glycogen type III from Sigma (St.
Louis, MO), and rabbit anti-mouse IgG from Miles (Naperville,
IL).
The hybridoma producing alR-3 was a kind gift from Dr.
Steven Jacobs, Burroughs-Welcome (Research Triangle Park,
NC). IgG was purified from mouse ascites fluid by sequential
ammonium sulfate precipitation and diethylaminoethyl (DEAE)Sephacel chromatography in 0.0175 M sodium phosphate
buffer. The preparations of alR-3 used in these studies were
homogeneous IgG as assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) in the presence of (8-mercaptoethanol. Fab'2 was prepared from alR-3
by pepsin digestion (28) and Fab', monovalent «IR-3 was
prepared from Fab'2 according to the method of Nisonoff et
al. (28).
Cells
HSF were derived from punch skin biopsies of the volar surface
of the forearm from normal adult volunteers. Cells were maintained in Dulbecco's Minimal Essential Medium (DME) containing 10% calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37 C in an atmosphere of 95% air, 5% CO2. Cells
were split 1:3 every 7-10 days and were used between the
seventh and 16th passage.
HEP G2 cells, a clonal human hepatoblastoma derived cell
line (29), were grown in the same media described above.
IGF-I Binding
125
I-IGF-I binding to HSF was performed to suspensions rather
than monolayers of HSF due to the presence of IGF binding
proteins adherent to the surface of HSF monolayers (20-22).
IGF-I was labeled by the fractional chloramine-T procedure
(30) to a specific activity of 150-300 /uCi/^g. HSF monolayer
cells were treated with 0.05% trypsin for 4 min after which a
3-fold excess of soybean trypsin inhibitor was added. 125I-IGFI was incubated with the cells for 3 h at 22 C after which cells
were pelleted in a Microfuge and counted for cell-associated
radioactivity.
Hep G2 cells were plated and grown in 16-mm multiwell
plates. They were washed three times with binding buffer.
After incubation with labeled IGF-I for 18 h at 4 C, plates were
washed three times with binding buffer, solubilized in 0.5 ml 1
N NaOH and 0.4-ml aliquots were counted. Although Hep G2
cells produce IGF binding proteins (31), these binding proteins
do not interfere with IGF-I binding to its receptor on Hep G2
monolayers in the same way as occurs on HSF monlayers
(32). All binding studies were performed in buffer of the following composition: 5 ITIM HEPES, 5 miwi KCI, 1 mM KH2PO4,1.2
HIM CaCI2-2H2O, 1 mM MgSO4, 288 mM sucrose. We previously have shown that substituting an isoosmotic concentration of sucrose for NaCI (288 mM) increases the apparent
affinity of IGF-I for its receptor and for the IGF binding protein
(manuscript in preparation).
MOL ENDO-1988
836
Amino Acid Transport in HSF
HSFs were plated as described previously (19), and the
method for transport of the nonmetabolizable amino acid, AIB
was adopted from Hollenberg and Cuatrecasas (33). On the
day of assay, cells were washed three times with Earle's
balanced salt solution (BSS), pH 7.4. After the incubation with
indicated concentrations of «IR-3 in BSS with 0.1% BSA for
1 h at 37 C, cells were washed once with BSS/BSA and
incubated with or without antimouse IgG for 2.5 h at 37 C.
Cells were also incubated with IGF-I separately as a positive
control. Unlabeled AIB (final concentration, 8 /ZM) and 0.2 fid
[3H]AIB were added and cells were incubated for 20 min at 37
C. Uptake was stopped by the addition of ice-cold PBS, pH
7.4. Plates were washed three times and then solubilized with
0.5 ml 1 N NaOH. Three hundred and fifty-microliter aliquots
were mixed with 3.5 ml Aquasol scintillation fluid (NEN) and
counted in a Searle Mark III Liquid Scintillation Counter. All
data points were performed in triplicate and were corrected
for protein content per well. Protein was measured with the
Bio-Rad (Richmond, CA) reagent using BSA as standard (34).
Glucose Incorporation into Glycogen in HEP G2
HEP G2 cells were plated in 16-mm multiwell trays in DME
(containing 1 mg/ml glucose) and 10% calf serum. After confluency, media was changed to serum-free DME and media
were changed twice daily for 2 days to ensure an adequate
glucose concentration for the cells. On the day of assay, the
cells were first incubated for 2 h at 37 C with fresh DME and
then were incubated for 1 h at 37 C with «IR-3. The cells were
washed once and then incubated with D-[U-14C]glucose (2 ^Ci/
well) and anti-mouse IgG or IGF-1 for 2 h at 37 C. Glycogen
was extracted and quantified by the method of Hofmann et al.
(35).
Statistical Analysis
Data are expressed as a percent of control values for each
incubation. Statistical significance was determined by the Student's t test for the mean of data points derived from at least
two experiments.
Acknowledgments
The authors wish to acknowledge the expert secretarial assistance of Ms. Terri Wiseman and Ms. Barbara Stock, and
the constructive comments and technical assistance of Patricia
Usher.
Received March 15,1988. Accepted May 23,1988.
Address requests for reprints to: Dr. Jeffrey S. Flier, Diabetes Unit, Beth Israel Hospital, 330 Brookline Avenue, Boston, Massachusetts 02215.
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16. Jacobs S, Cook S, Svoboda ME, Van Wyk JJ 1986
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17. Van Wyk JJ, Graves DC, Casella J, Jacobs S 1986
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18. Flier JS, Usher P, Moses AC 1986 Monoclonal antibody
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