From the
Transgenic mice which overexpress kinase-deficient human insulin
receptors in muscle were used to study the relationship between insulin
receptor tyrosine kinase and the in vivo activation of several
downstream signaling pathways. Intravenous insulin stimulated insulin
receptor tyrosine kinase activity by 7-fold in control muscle
versus
Insulin's diverse actions are initiated by binding to its
specific transmembrane receptor. The intracellular
Activation
of the insulin receptor and other growth factor-activated receptor
tyrosine kinases including the insulin-like growth factor 1
(IGF-1)
Previous
studies have demonstrated that kinase-deficient insulin receptors when
overexpressed in cultured cells have an impaired ability to mediate
IRS-1 phosphorylation, PI 3-kinase activation, and activation of the
ras-MAP kinase
cascade
(1, 2, 4, 11, 18, 19, 20, 21) .
However, the relationship between the receptor tyrosine kinase and
activation of these (or other) cellular signaling intermediates in any
in vivo target tissues for insulin has not been directly
addressed. We have recently generated lines of transgenic mice which
express a dominant-negative mutant human insulin receptor (Ala
In the present study,
we investigated the consequences of impaired insulin receptor kinase
activity for the ability of insulin administered in vivo to
regulate molecules that participate in several important signaling
pathways (IRS-1, PI 3-kinase, MAP kinase, and c-fos mRNA) in
muscle. We also provide evidence which supports the hypothesis that the
mechanism(s) responsible for trans-dominant effects of kinase-deficient
insulin receptor expression involves the formation of hybrid
heterodimer complexes between endogenous murine receptors and
overexpressed mutant human receptors.
Muscle lysate containing 3 mg of solubilized protein
was incubated with
Skeletal muscle is a key site for insulin-mediated in
vivo glucose disposal
(29) . Furthermore, insulin- or IGF-1
receptor-mediated cellular signaling plays a major role in normal
muscle differentiation and development
(30) , promotes amino acid
uptake (31), and inhibits muscle proteolysis
(32) . In addition,
impaired insulin stimulation of muscle insulin receptor tyrosine kinase
activity has been demonstrated in humans with insulin-resistant
non-insulin-dependent diabetes mellitus and in similarly affected
rodent model systems
(33, 34) . The present studies used
an in vivo model to directly investigate the consequences of
impaired skeletal muscle insulin receptor tyrosine kinase activity on
downstream insulin signaling events in this important target tissue.
We previously demonstrated that transfected mutant Thr
In contrast to the impairment of receptor
autophosphorylation which we observed in transgenic muscle,
overexpression of human insulin receptors with mutated ATP-binding
sites (A/K 1018) in rodent fibroblasts
(35) or cultured
adipocytes
(36) reportedly did not reduce the net amount of
insulin-stimulated receptor autophosphorylation. The reason for this
discrepancy is not clear although it might relate to differences in the
ability of mutant receptor
Studies performed in cultured cell systems have suggested that
normal insulin-mediated receptor autophosphorylation and tyrosine
kinase activity is required for insulin-stimulated phosphorylation of
IRS-1 and activation of PI
3-kinase
(1, 18, 19, 20) . Furthermore,
in obese insulin-resistant mice, reduced insulin receptor
autophosphorylation was associated with similarly reduced IRS-1
phosphorylation
(41) . However, glucocorticoid treatment of rats
was reported to impair insulin receptor tyrosine kinase activity
without an apparent reduction in IRS-1 phosphorylation
(42) .
Importantly, our results clearly show that the severe reduction in
maximal insulin-stimulated receptor tyrosine kinase activity (toward
poly-Glu-Tyr) in transgenic muscles resulted in proportional defects
involving in vivo insulin-stimulated IRS-1 tyrosyl
phosphorylation and insulin-stimulated PI 3-kinase activity in IRS-1
immunoprecipitates. This is consistent with results recently reported
by Wilden and Kahn
(20) who studied these effects of insulin
using different insulin receptor mutants with progressive tyrosine
kinase defects which were expressed in CHO cells. Experiments that we
performed with control mice also showed that the insulin dose-response
for receptor kinase, IRS-1 phosphorylation, and PI 3-kinase were nearly
identical. These data confirm findings recently observed in the rat
(43) and with isolated mouse soleus muscles
(44) . The
ability of kinase-deficient human receptors to impair muscle IRS-1 or
PI 3-kinase activation mediated by endogenous mouse receptors is
consistent with the observation that kinase-deficient insulin receptors
transfected into cultured cells inhibit the ability of
endogenous
(35, 36) or co-expressed
(38) normal
insulin receptors to mediate trans-phosphorylation of IRS-1.
Like
other growth factors, insulin stimulates the activation of numerous
cellular Ser/Thr kinases including members of the MAP kinase (or ERK)
family. Current evidence suggests that an important role of MAP kinases
in growth factor or insulin signaling involves regulation of both cell
proliferation (G
We and
others previously suggested that kinase-deficient insulin receptors
expressed in transfected cells are unable to promote MAP kinase
activation
(11, 20) . However, these results were based
on phosphorylation of myelin basic protein or MAP-2 by total cell
lysates. Furthermore, no information is available concerning the
relationship between the receptor kinase and c-fos gene
induction. Interestingly, kinase-negative EGF receptor mutants have
been reported to be capable of normally activating MAP kinase
(49) and c-fos gene expression
(50) . By using an
electrophoretic mobility assay, we determined that insulin stimulated
the phosphorylation of p42 (and p44) MAP kinase in a time- and
dose-dependent manner in normal muscle. The ability of insulin to
induce c-fos gene expression in control mouse muscle was
similar to results recently reported by Olson and Pessin (26) using rat
adipose and cardiac tissue. The fact that insulin-stimulated p42 MAP
kinase phosphorylation and c-fos mRNA accumulation in
transgenic muscle were markedly impaired demonstrates that these
effects are dependent upon the receptor tyrosine kinase in this
important target tissue.
Since a number of patients with the
phenotype of severe insulin are simple heterozygotes for insulin
receptor alleles which encode kinase-deficient receptor
mutants
(51) , it is important to characterize the mechanisms
responsible for the dominant-negative properties of such receptor
variants. Mutant forms of several other receptor tyrosine kinases
including EGF
(52) , platelet-derived growth factor
(53) ,
fibroblast growth factor
(54) , and keratinocyte growth factor
(55) receptors also possess dominant-negative properties. The
formation of nonfunctional heterodimers with wild type receptors has
been demonstrated in several of these
cases
(52, 53, 54) . In the case of the insulin
receptor, hybrid
In the present study, we
demonstrated that the number of residual mouse insulin receptors
present in solubilized muscle proteins from transgenic mice after
immunodepletion of human insulin receptors was substantially lower than
the level of endogenous receptors in control muscle analyzed under the
same conditions. These results provide indirect evidence that mouse
insulin receptors formed hybrid receptor complexes with mutant human
receptors in vivo. Alternatively, it is also possible that the
level of endogenous muscle insulin receptor expression in transgenic
mice was lower as a consequence of forced overexpression of human
receptors.
Using a similar immunoprecipitation approach, we were
able to determine that only a small proportion of endogenous IGF-1
receptors could be isolated from transgenic muscle by the
human-specific anti-insulin receptor antibody. This may serve to
explain the fact that low dose IGF-1 was able to provoke
IRS-1-associated PI 3-kinase activation in transgenic muscle to a
degree that was similar to that observed in control muscle. Thus, the
lack of substantial mutant insulin-IGF-1 receptor hybrids was
associated with the absence of transdominant inhibition of an effect
mediated by endogenous IGF-1 receptors. In contrast, overexpression of
A/K 1018 insulin receptors in rat 1 fibroblasts was reportedly
associated with impairment of IGF-1-mediated IRS-1 phosphorylation and
mitogenesis
(57) . Since we have observed that overexpression of
human insulin receptors in CHO cells is associated with substantial
formation of hybrids with endogenous IGF-1 receptors (58), it is
logical to conclude that the stoichiometry of transfected insulin
receptors (
In
summary, we have used a transgenic mouse model characterized by
overexpression of tyrosine kinase-deficient human insulin receptors in
muscle to determine that activation of the insulin receptor tyrosine
kinase is required for stimulation of several known insulin signaling
pathways in this tissue. The presence of mutant human insulin receptors
exerts dominant-negative effects at the level insulin-stimulated
receptor autophosphorylation and tyrosine kinase activation.
Furthermore, our findings suggest that the formation of hybrid
mutant-normal receptor heterodimers contributes to mechanisms which
underlie the dominant-inhibitory effects of mutant receptor expression
for in vivo insulin signaling.
We are grateful for the technical assistance of Luigi
Gnudi (Beth Israel Hospital, Boston), for advice provided by Harald H.
Klein (University of Lubeck, Lubeck, Germany), and for valuable
reagents provided by Robert J. Smith, Bentley Cheatham, C. Ronald Kahn,
and Morris White (Joslin Diabetes Center, Boston); Ken Siddle
(Cambridge University, UK), John Blenis (Harvard Medical School),
Richard Roth (Stanford University, Palo Alto, CA), and Lu-Hai Wang
(Mount Sinai, New York, NY).
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1.5-fold in muscle from transgenic mice. Similarly,
insulin failed to stimulate tyrosyl phosphorylation of receptor
-subunits or insulin receptor substrate 1 (IRS-1) in transgenic
muscle. Insulin substantially stimulated IRS-1-associated
phosphatidylinositol (PI) 3-kinase in control versus absent
stimulation in transgenic muscles. In contrast, insulin-like growth
factor 1 modestly stimulated PI 3-kinase in both control and transgenic
muscle. The effects of insulin to stimulate p42 mitogen-activated
protein kinase and c-fos mRNA expression were also markedly
impaired in transgenic muscle. Specific immunoprecipitation of human
receptors followed by measurement of residual insulin receptors
suggested the presence of hybrid mouse-human heterodimers. In contrast,
negligible hybrid formation involving insulin-like growth factor 1
receptors was evident. We conclude that (i) transgenic expression of
kinase-defective insulin receptors exerts dominant-negative effects at
the level of receptor autophosphorylation and kinase activation; (ii)
insulin receptor tyrosine kinase activity is required for in vivo insulin-stimulated IRS-1 phosphorylation, IRS-1-associated PI
3-kinase activation, phosphorylation of mitogen-activated protein
kinase, and c-fos gene induction in skeletal muscle; (iii)
hybrid receptor formation is likely to contribute to the in vivo dominant-negative effects of kinase-defective receptor expression.
-subunits of
the insulin receptor possess intrinsic protein tyrosine kinase activity
which is stimulated by insulin binding to the extracellular
-subunits and is augmented by
-subunit tyrosyl
autophosphorylation. The preponderance of current data suggests that
most, if not all, of insulin's biological effects are mediated by
the insulin receptor tyrosine kinase
(1) . However, several
mutated versions of the insulin receptor with impaired tyrosine kinase
activity (toward exogenous substrates) reportedly retain the capacity
to mediate signaling toward one or more biological actions of
insulin
(2, 3, 4, 5, 6) . In
addition, certain monoclonal anti-insulin receptor antibodies have been
reported to mimic the actions of insulin without activation of the
receptor tyrosine kinase
(7, 8) , although these findings
have subsequently been questioned by others
(9) .
(
)
receptor is associated with stimulation
of several common intracellular signaling pathways. Specific
intermediates that participate in these pathways include
phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, p21 ras,
and mitogen-activated protein (MAP)
kinases
(1, 10, 11, 12) .
Receptor-mediated tyrosyl phosphorylation of a major 185-kDa insulin
receptor substrate, IRS-1, plays an important role in mediating these
insulin signaling pathways since phosphorylated IRS-1 binds to a number
of Src homology 2 (SH2) domain-containing proteins including the p85
subunit of PI 3-kinase, GRB-2, nck, and SH-PTP2
(syp)
(1, 13, 14) . In addition, recent
studies have shown that insulin can activate both PI 3-kinase and the
ras-MAP kinase cascade in mice lacking
IRS-1
(15, 16) . This IRS-1-independent signaling may
involve receptor-mediated phosphorylation of Shc
(17) or a newly
identified substrate now referred to as IRS-2
(16) .
Thr) in skeletal and cardiac muscle
(22) . These
mice are mildly insulin resistant as determined by hyperinsulinemia and
reduced glycemic responsiveness to exogenous insulin
(22) .
Furthermore, skeletal muscle from these transgenic mice displayed
reduced insulin-stimulated insulin receptor tyrosine kinase
activity
(22) . Therefore, this animal model provides a unique
opportunity to study the effects of altered insulin receptor tyrosine
kinase in an important insulin target tissue.
Transgenic Mice
The construction of transgenic
mice with muscle-specific overexpression of a mutant human insulin
receptor (Ala
Thr) was described
previously
(22) . Mice (inbred FVB-NJ strain) used in the present
study were 8-12-week-old hemizygous transgenics derived from two
independent lines of transgenic mice. Similar numbers of mice from both
lines were used for experiments described below. Age- and sex-matched
littermate control mice were used for each experiment.
Assessment of Muscle Insulin Receptor
Expression
The level of muscle insulin receptor expression in
control versus transgenic mice was determined by
immunoblotting with a nonspecies-specific anti-insulin receptor
antibody (IR-C) directed against the C terminus of the receptor
-subunit (kindly provided by B. Cheatham and C. R. Kahn, Joslin
Diabetes Center, Boston). Muscle protein lysates were prepared from
samples of powdered frozen muscle as described below. Protein
concentration was determined using the Bradford dye binding assay kit
(Bio-Rad) or BCA protein assay reagent (Pierce). Aliquots of muscle
lysate containing solubilized protein (300 µg) were separated by
SDS-polyacrylamide gel electrophoresis, followed by transfer to
nitrocellulose membranes. Membranes were blocked with 5% nonfat dried
milk in TNA (20 mM Tris-HCl, pH 7.8, 150 mM NaCl,
0.01% sodium azide) with the addition of 0.05% Tween 20 for 2 h at room
temperature followed by the addition of the IR-C antibody (1:500) for
an additional 2 h. After removal of unbound antibody, membranes were
incubated with horseradish peroxidase-conjugated anti-rabbit IgG for 1
h followed by washing for 2
10 min in TNA plus 0.05% Nonidet
P-40 and 2
10 min in TNA with 0.1% Tween 20. Detection was
accomplished using enhanced chemiluminescence (Amersham).
Measurement of Skeletal Muscle Insulin Receptor Tyrosine
Kinase Activity
The procedure for measurement of muscle insulin
receptor tyrosine kinase activity was similar to previously described
methods (22, 23). In brief, fasted mice (overnight) were anesthetized
with Avertin (Tribromoethanol, tertamyl alcohol, Aldrich) and kept on a
warming pad. Samples of gluteal or gastrocnemius muscle (50-200
mg) were removed 2 min following intravenous injection (tail vein) of
the indicated doses of insulin or recombinant IGF-1 (Genentech, South
San Francisco, CA) or saline. Muscle samples were snap-frozen in liquid
nitrogen and Polytron homogenized in ice-cold Buffer A containing 50
mM HEPES, pH 7.5, 150 mM NaCl, 10 mM sodium
pyrophosphate, 2 mM NaVO
, 1
mM MgCl
, 1 mM CaCl
, 10
mM NaF, 2 mM EDTA, 2 mM phenylmethylsulfonyl
fluoride, 5 µg/ml leupeptin, 1% Nonidet P-40, and 10% glycerol.
After incubation for 30 min and brief centrifugation, supernatant
containing 100 µg of soluble protein was incubated overnight at 4
°C in microtiter wells coated with a nonspecies-specific insulin
receptor antibody (AB-3, Oncogene Science). Tyrosine kinase activity of
the bound insulin receptors was measured by adding 20 µl of
poly-Glu-tyr (4:1) (Sigma) (4 mg/ml in water) and 20 µl of reaction
mixture containing 10 mM MgCl
, 5 mM
MnCl
, 0.5 µM ATP, and 7 µCi of
[
-
P]ATP. After 20 min of incubation at 20
°C, the reaction was stopped by spotting onto filter paper (Whatman
3MM). Filter papers were washed with 10% trichloroacetic acid and 10
mM sodium pyrophosphate. Radioactivity was determined by
Cerenkov counting.
Tyrosyl Phosphorylation of Insulin Receptor and
IRS-1
Muscle lysates were prepared as described above.
Solubilized proteins were resolved by SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose membranes. Membranes
were blocked with TNA containing 5% albumin for 2 h at 37 °C. The
membranes were then incubated with affinity-purified
anti-phosphotyrosine antibody (PY provided by R. J. Smith, Joslin
Diabetes Center, 24) in TNA plus 5% bovine serum albumin for
12-16 h at 4 °C. Membranes were washed 2
5 min in TNA
plus 0.05% Nonidet P-40 and 1
5 min in TNA plus 0.1% Tween 20.
Bound antibodies were detected by incubation with
I-Protein A (1 µCi/ml) (ICN Biomedical, Costa Mesa,
CA) for 1 h at room temperature. Bands corresponding to phosphorylated
IRS-1 or insulin receptor
-subunit were quantitated using a
Molecular Dynamics PhosphorImager. The level of IRS-1 protein
expression was assessed by immunoblotting of mouse muscle lysate with
anti-IRS-1 antibody (gift of Morris White, Joslin Diabetes Center)
using methods described above.
Measurement of Muscle PI 3-kinase Activity
Gluteal
and gastrocnemius muscle lysates were prepared as described above after
2 min intravenous stimulation with insulin, IGF-1, or saline. Cleared
muscle lysates were assayed for PI 3-kinase activity that was measured
in immunoprecipitates obtained with antibodies to IRS-1 (IRS-1,
affinity purified polyclonal antibody prepared by injecting rabbits
with a synthetic peptide corresponding to the last 14 amino acids in
the C-terminal region of rat IRS-1, provided by R. J. Smith, Joslin
Diabetes Center). Enzyme activity in reconstituted immunoprecipitates
was assayed as described previously
(25) , with some
modification.
IRS-1 and protein A-Sepharose (Pierce). Immune
complexes were washed three times with phosphate-buffered saline
containing 1% Nonidet P-40 and 100 µM
Na
VO
, three times with 100 mM
Tris-HCl, pH 7.5, containing 500 mM LiCl, and twice with 10
mM Tris-HCl, pH 7.5, containing 0.1 M NaCl, 1
mM EDTA, and 100 µM Na
VO
.
The pellets were resuspended in 50 µl of the Tris-NaCl buffer
containing 12 mM MgCl
and 10 µg of
phosphatidylinositol (Avanti Polar Lipids Inc., Alabaster, AL). The PI
3-kinase reaction (room temperature) was initiated by adding 10 µl
of 440 µM ATP containing 30 µCi of
[
P]ATP. After 10 min of vigorous vortexing,
the reaction was stopped by the addition of 20 µl of 8 N
HCl and 160 µl of chloroform/methanol (1:1, v/v). The phases were
separated by centrifugation, and the lower organic phase was spotted
onto thin layer chromatography plates
(25) . Lipids were resolved
by chromatography in
CH
OH/CHCl
/H
O/NH
OH
(60:47:11.3:2) and visualized by autoradiography. Radioactivity in the
spots which co-migrated with a PI-3-P standard was quantitated using a
PhosphorImager.
Assessment of p42/p44 Map Kinase
Phosphorylation
Mice were fasted for 48 h, followed by
anesthesia with Avertin and placement on a warming pad. Following
intravenous administration of the indicated insulin doses or saline,
mice were sacrificed at the indicated time points and muscle was
removed and snap-frozen in liquid nitrogen. Muscle was homogenized and
solubilized in a buffer containing 20 mM HEPES, pH 7.0, 5
mM EDTA, 10 mM EGTA, 10 mM MgCl,
50 mM
-glycerophosphate, 1 mM
Na
VO
, 2 mM dithiothreitol, 2 µg/ml
leupeptin, 5 µg/ml aprotinin, 40 µg/ml phenylmethylsulfonyl
fluoride, and 1% Nonidet P-40 followed by incubation for 30 min at 4
°C, and brief centrifugation. The phosphorylation state of p42 and
p44 MAP kinases was assessed by electrophoretic mobility as follows.
Aliquots of muscle lysate containing 70 µg of solubilized protein
were separated by 10% SDS-polyacrylamide gel electrophoresis followed
by electrophoretic transfer to nitrocellulose membranes. Membranes were
probed with an anti-ERK1/ERK2 antibody (
-C2, provided by John
Blenis, Harvard Medical School). The conditions for immunoblotting,
washing, and detection by enhanced chemiluminescence are described
above.
Measurement of c-fos mRNA
Expression
For c-fos mRNA expression, fasted mice
(overnight) were anesthetized with Avertin, followed by intraperitoneal
injection of 10 milliunits/g insulin as a combination of regular
(Humulin R, Lilly) and long-acting (Humulin N) insulin
(26) . A
dose of intraperitoneal glucose (1.0 mg/g body weight) that was
empirically determined to prevent the development of hypoglycemia was
also administered. After 1 h, mice were sacrificed, and a sample of
muscle was removed and snap-frozen in liquid nitrogen. Twenty µg of
total RNA
(27) was separated by electrophoresis on 1.2%
formaldehyde-agarose gels followed by transfer to nylon membranes.
Membranes were hybridized
(22) with a random-primed
[P]dCTP-labeled fragment of rat c-fos cDNA (provided by M. Jakubowski, Beth Israel Hospital, Boston).
After washing with high stringency
(28) , membranes were
subjected to autoradiography as described previously
(28) . A
random-primed labeled [
P]dCTP
-actin cDNA
probe was also prepared and used as described previously
(28) .
The bands corresponding to c-fos and
-actin transcripts
were quantitated by PhosphorImager.
Insulin Receptor Immunoprecipitation: Assessment of
Potential Hybrid Receptor Formation
Muscle lysates from control
and transgenic mice were prepared using Buffer A as described above.
Aliquots of lysate (500 µl) containing 200 µg of solubilized
protein were immunoprecipitated with a monoclonal human-specific
anti-insulin receptor antibody (83-14, kindly provided by K.
Siddle, Cambridge University, U.K.) and Protein A-Trisacryl beads
(Pierce) for 2 h at 4 °C. After immunoprecipitating three times,
aliquots of the supernatant were used for an insulin receptor binding
assay and immunoblotting with nonspecies-specific anti-insulin receptor
antibody (IR-C). Immunoprecipitated proteins (present in the pellet)
were also analyzed by immunoblotting with IR-C. For the insulin binding
assay, procedures were as described previously
(22) . For Western
blotting of insulin receptors present in the pellet or supernatant,
proteins were separated, blotted, and probed with IR-C exactly as
described above (``Quantitation of Muscle Insulin Receptor
Expression''). IGF-1 receptors present in solubilized muscle
lysates (and in 83-14 immunoprecipitates from muscle lysates)
were detected by immunoblotting with a polyclonal anti-mouse IGF-I
receptor antibody (-IGFR, provided by L. H. Wang, Mount Sinai, New
York). Protein lysates prepared from CHO cells which overexpress human
IGF-I receptors (CHO-IGFR, provided by Richard Roth, Stanford
University) and CHO cells which overexpress human insulin receptors
(CHO-IR, 4) were used as controls in the above experiments.
Overexpression of Mutant Human Insulin Receptors in
Transgenic Mice
Two independent lines of transgenic mice which
overexpress mutant Thr human insulin receptors in
skeletal and cardiac muscle were generated and characterized as
described previously
(22) . In order to verify that mutant human
receptors were overexpressed in muscles that were subsequently studied,
solubilized gluteal and gastrocnemius muscle proteins were separated by
SDS-PAGE followed by immunoblotting with antibody IR-C which recognizes
both murine and human insulin receptors. As shown in Fig. 1,
analysis of these muscles confirmed that the level of mutant receptor
expression in both lines of transgenic mice was severalfold higher than
the expression of endogenous insulin receptors in control mice.
Figure 1:
Overexpression of mutant human insulin
receptors in skeletal muscle of transgenic mice. Solubilized muscle
proteins (300 µg) prepared from control and transgenic mice were
fractionated by 7% SDS-PAGE followed by immunoblotting with a
non-species-specific anti-insulin receptor antibody. This example shows
results obtained with gluteal muscle derived from three control and
three transgenic mice. The arrow indicates a 95 kDa protein
band which corresponds to the insulin receptor
-subunit.
Insulin Receptor Tyrosine Kinase Activity Following in
Vivo Insulin Stimulation
We previously showed that gluteal
muscle insulin receptor tyrosine kinase activity was markedly impaired
in transgenic mice after administration of insulin by intraperitoneal
injection
(22) . In the present study, insulin was administered
intravenously followed by removal of gluteal or gastrocnemius muscle
samples. Equal aliquots of solubilized muscle proteins were applied to
microwells coated with the nonspecies-specific insulin receptor
antibody (AB-3), and in vitro insulin receptor kinase activity
toward poly-Glu-Tyr was measured. Experiments conducted in control mice
established that maximal insulin receptor kinase activity was achieved
with insulin doses greater than 5 milliunits/g
(Fig. 2A). Thus, subsequent experiments were performed
using this maximally effective insulin dose. Fig. 2B shows that insulin stimulated receptor phosphotransferase activity
by 5-6-fold over basal in control gluteal and gastrocnemius
muscles. In contrast, insulin-stimulated receptor tyrosine kinase
activity in muscles from transgenic mice was markedly impaired (less
than 1-fold over basal).
Figure 2:
Insulin receptor tyrosine kinase activity
after in vivo insulin stimulation. Tyrosine kinase activity
toward poly-Glu-Tyr was determined using gluteal or gastrocnemius
muscles obtained 2 min after intravenous insulin (or saline) injection.
A, dose-response curve generated using intravenous injection
of increasing insulin doses into control mice. Each point represents
mean ± S.E. of results obtained with eight mice. Data are
expressed as nanomoles of ATP transferred to the substrate per
milligram of gluteal muscle protein. B, insulin receptor
tyrosine kinase activity obtained using muscle from control
(C) and transgenic (TG) mice with (+) or without
(-) in vivo insulin stimulation (5 milliunits/g body
weight). The results are expressed as percentage of basal control and
represent mean ± S.E. of data from two experiments (n = 6 controls, n = 6 transgenic mice in each
experiment). * = transgenic versus insulin-stimulated
control gluteal muscle, p < 0.0001. ** = transgenic
versus insulin-stimulated control gastrocnemius muscle, p < 0.01).
Effect of the Transgene on Insulin-stimulated Receptor
and IRS-1 Tyrosyl Phosphorylation
Tyrosyl phosphorylation of
insulin receptor -subunits and IRS-1 was assessed by direct
immunoblotting of solubilized muscle proteins with antiphosphotyrosine
antibody. First, dose-response curves were generated using gluteal
muscle derived from control mice (Fig. 3). For both insulin
receptor and IRS-1 phosphorylation, the results paralleled the effect
of insulin to stimulate receptor tyrosine kinase activity toward the
exogenous substrate since partial stimulation was evident with 1.0
milliunits/g and maximal stimulation was achieved with 5.0-50
milliunits/g. For subsequent experiments 5 milliunits/g insulin was
used.
Figure 3:
Tyrosyl phosphorylation of insulin
receptor and IRS-1: insulin dose-response. Gluteal muscles were
obtained from control mice after injection of saline or increasing
insulin doses. Muscle proteins were resolved by SDS-PAGE followed by
immunoblotting with anti-phosphotyrosine antibody. Tyrosyl
phosphorylated proteins detected using I-Protein A were
quantitated by PhosphorImager analysis. A, dose-response curve
for insulin-stimulated receptor
-subunit phosphorylation in
control mice. B, dose-response curve for insulin-stimulated
IRS-1 tyrosyl phosphorylation in control mice. Each point represents
mean ± S.E. of data obtained from four
mice.
Insulin increased receptor autophosphorylation by 200% in
gluteal and 182% of basal in gastrocnemius muscles from control mice
(Fig. 4, A and B). Similarly, insulin
stimulated IRS-1 phosphorylation by 297% in control gluteal and 208% of
basal in control gastrocnemius muscles (Fig. 4, A and
C). In contrast, in both sets of muscle derived from
transgenic mice there was no detectable insulin stimulation of either
receptor or IRS-1 tyrosyl phosphorylation (Fig. 4). In separate
experiments we determined that the level of IRS-1 protein expression in
transgenic gluteal muscle was comparable to the level of expression in
control mice (not shown).
Figure 4:
Effect
of mutant insulin receptor transgene on receptor and IRS-1 tyrosyl
phosphorylation. A, this example autoradiogram shows basal and
maximal insulin-stimulated phosphorylation of IRS-1 (185 kDa band) and
insulin receptor -subunit (95 kDa band) in control (C)
and transgenic (TG) gluteal muscles. B, quantitation
of insulin receptor autophosphorylation in gluteal and gastrocnemius
muscles following saline (-) or 5 milliunits/g insulin (+)
injection. The results represent the mean ± S.E. of data
obtained in three separate experiments (n = 6 controls,
n = 6 transgenic mice in each experiment) and are
expressed as percentage of basal control. * = transgenic
versus insulin-stimulated control gluteal muscle, p < 0.005. ** = transgenic versus insulin-stimulated control gastrocnemius muscle, p <
0.05. C, quantitation of IRS-1 phosphorylation. Results
represent mean ± S.E. of data obtained in three separate
experiments (n = 6 control, n = 6
transgenic mice in each experiment). * = transgenic versus insulin-stimulated control gluteal muscle, p < 0.0001.
** = transgenic versus insulin-stimulated control
gastrocnemius muscle, p < 0.001.
Effect of the Transgene on Insulin and IGF-1-stimulated
PI 3-Kinase Activity
To investigate whether the defect involving
insulin-stimulated IRS-1 phosphorylation in transgenic muscle was
associated with impairment of PI 3-kinase activation, PI 3-kinase
activity in anti-IRS-1 immunoprecipitates was assayed. Initially, we
found that the in vivo insulin dose-response curve for PI
3-kinase in control muscle paralleled insulin's effects on
receptor kinase activity or IRS-1 phosphorylation
(Fig. 5A). Subsequently, we compared control muscles to
transgenic muscles after treatment with 5 milliunits/g intravenous
insulin. PI 3-kinase activity in control gluteal and gastrocnemius
muscles was increased by 37- and 16-fold of basal, respectively
(Fig. 5, B and C). In contrast, there was no
detectable insulin stimulation of PI 3-kinase activity in transgenic
gluteal and gastrocnemius muscles (Fig. 5, B and
C).
Figure 5:
Insulin-stimulated PI-3-kinase activity.
Muscle proteins obtained from insulin, or saline-treated mice were
immunoprecipitated with IRS-1 antibody. PI 3-kinase activity in
immunoprecipitates was assayed by measurement of P
incorporation into PI-3-P after thin layer chromatography. A,
dose-response curve generated by injection of increasing insulin doses
into control mice. Each point represents mean ± S.E. of results
obtained from four mice. B, this example autoradiogram shows
basal and insulin-stimulated PI-3-P phosphorylation in anti-IRS-1
immunoprecipitates from control (C) and transgenic
(TG) gluteal muscle. C, quantitation of PI-3-kinase
activity in gluteal and gastrocnemius muscles from mice treated with
saline (-) or 5 milliunits/g insulin (+). The results
represent mean ± S.E. of data obtained in three separate
experiments (n = 6 control mice, n = 6
transgenic mice in each experiment). * = transgenic versus insulin-stimulated control gluteal muscle, p < 0.02.
** = transgenic versus insulin-stimulated control
gastrocnemius muscle, p < 0.005.
In order to determine whether overexpression of mutant
insulin receptors would exert transdominant effects on IGF-1
receptor-mediated signaling, we measured muscle PI 3-kinase activation
after in vivo IGF-1 administration. We first established that
intravenous injection of IGF-1 was capable of exerting a biologic
response. Thus, doses of 0.5 µg/g body weight or more provoked
hypoglycemia in control mice. Using a high dose of IGF-1 (2.0
µg/g), PI 3-kinase activity in control mouse muscle was
substantially augmented (974 ± 193% of basal, n = 7 mice). In contrast, stimulated transgenic muscle PI
3-kinase activity was only 231 ± 33% of basal (n = 8 mice). However, we found that this dose of IGF-1 was
associated with persistent activation of muscle insulin receptor
tyrosine kinase in control mice (not shown). Therefore, in subsequent
experiments we used 0.5 µg/g of IGF-1, a dose that was not
associated with activation of muscle insulin receptors. This lower
IGF-1 dose resulted in a similar degree of modest PI 3-kinase
stimulation in both control and transgenic muscle (274.5 ± 46%
of basal versus 244 ± 33%, respectively, not
significant). Thus, the ability of IGF-1 to stimulate PI 3-kinase
exclusively through its own receptors was apparently preserved in
transgenic muscle.
Insulin Stimulation of Muscle MAP Kinase
Phosphorylation
In order to explore the effect of impaired
muscle insulin receptor tyrosine kinase activity on the ability of
insulin to activate MAP kinases, we used an electrophoretic mobility
shift assay to assess the phosphorylation state of p42 (ERK2) and p44
(ERK1) MAP kinases. Preliminary experiments showed that maximal MAP
kinase phosphorylation occurred with insulin doses 10 milliunits/g
(not shown). As depicted in Fig. 6A, insulin
administration to control mice resulted in time-dependent
phosphorylation of muscle p42 MAP kinase with maximal effects occurring
by 5 min. This time- and dose-dependent phosphorylation was also seen
with p44 MAP kinase, although this isoform is apparently not as
abundantly expressed in skeletal muscle. In comparing the molecular
weight shift of MAP kinase in gluteal muscles derived from transgenic
versus control mice, we noted that insulin stimulated
substantial phosphorylation of p42 MAP kinase in control muscle with
absent or minimal stimulation of a p42 MAP kinase-phosphorylated band
shift in muscle from transgenic mice (Fig. 6B).
Figure 6:
Insulin-stimulated phosphorylation of
MAP kinase. After intravenous injection of saline or 10 milliunits/g
insulin, muscle proteins were resolved by 10% SDS-PAGE followed by
immunoblotting with anti-MAP kinase antibody. Bands corresponding to
dephosphorylated (lower) and phosphorylated (upper)
p42 and p44 MAP kinase were detected using enhanced chemiluminescence.
A, time course generated by obtaining gluteal muscle samples
from five different mice at the indicated time points after insulin
administration. B, comparison of saline (-) and
insulin-stimulated (+) MAP kinase phosphorylation in control
(C) versus transgenic (TG) gluteal muscle
obtained 5 min after injection. This example shows results obtained
with two control and two transgenic mice. Similar results were obtained
with seven additional mice from each group.
Insulin Stimulation of c-fos mRNA Expression
To
study whether the marked impairment of muscle insulin receptor kinase
in transgenic mice would affect insulin's ability to stimulate
the expression of immediate early genes, we assessed the level of
c-fos mRNA by Northern blot analysis after in vivo insulin stimulation. Preliminary experiments demonstrated that
c-fos mRNA levels in muscle from control mice were maximally
elevated 1 h after intraperitoneal insulin administration and returned
to basal levels by 6 h, while -actin mRNA levels remained constant
(not shown). In addition, we determined the dose-response
characteristics of insulin-stimulated c-fos mRNA expression.
As shown in Fig. 7A, maximal stimulation of c-fos expression was achieved with insulin doses of 10 milliunits/g or
greater. When transgenic and control mice were treated with 10
milliunits/g of insulin, the level of c-fos mRNA expression in
gluteal muscle was 664 ± 118% of basal in control mice
versus 276 ± 56% in transgenics
(Fig. 7B). Similar impairment of insulin-stimulated
c-fos expression in transgenic mice was observed using
gastrocnemius muscles, although the degree of stimulation in control
mice was less in this muscle (Fig. 7B).
Figure 7:
Insulin-stimulated induction of c-fos mRNA expression. Sixty min after intraperitoneal injections of
saline or insulin (and glucose), muscle was excised and total RNA was
prepared. A, dose-response curve generated using increasing
insulin doses administered to control mice. Each point represents mean
± S.E. of 3 mice; the relative level of c-fos mRNA was
determined by Northern blotting followed by PhosphorImager quantitation
and is expressed as a percentage of the basal c-fos mRNA
level. B, quantitation of insulin-stimulated c-fos mRNA expression in muscle from control (C) and transgenic
(TG) mice with (+) or without (-) in vivo insulin stimulation (10 milliunits/g). The results are expressed
as percentage of basal control and represent mean ± S.E. of data
from three separate experiments (n = 8 controls, n = 8 transgenic mice in each experiment). * =
transgenic versus insulin-stimulated control gluteal muscle,
p < 0.0005. ** = transgenic versus insulin-stimulated control gastrocnemius muscle, p <
0.01.
Assessment of Potential Hybrid Receptor
Formation
To explore potential mechanisms for the
dominant-negative effects of mutant receptor expression, we
investigated whether hybrid receptors composed of mouse-human insulin
receptor halves or mouse IGF-1 receptor-human insulin receptor halves
were present in muscle from transgenic mice. Since no mouse-specific
anti-insulin receptor antibody exists, we could only indirectly address
the possible formation of hybrid mouse-human insulin receptors. Muscle
protein lysates from control and transgenic mice were
immunoprecipitated with a human-specific monoclonal anti-insulin
receptor antibody) followed by measurement of total insulin receptors
remaining in the supernatant and receptors present in the pellet.
Preliminary experiments established that three rounds of
immunoprecipitation were sufficient to deplete more than 95% of human
receptors present in lysates from transgenic muscle (not shown). As
shown in Fig. 8A, 83-14 immunoprecipitated human
receptors from transgenic muscle but failed to immunoprecipitate mouse
insulin receptors from control muscle lysate as expected. After
immunoprecipitation with 83-14, less insulin receptors (mouse)
remained in the supernatants from transgenic muscle compared with
supernatants from control muscle lysates exposed to the same
conditions. The reduced abundance of mouse insulin receptors in
transgenic muscle after depletion of human receptor protein was
determined by immunoblotting (Fig. 8B) and by insulin
binding assays performed after immobilizing insulin receptors in
post-immunoprecipitation lysates on microtiter wells coated with the
nonspecies-specific insulin receptor antibody (Fig. 8C).
Thus, specific insulin binding in transgenic lysates was reduced to
0.35 ± 0.1 pmol of I-insulin bound/mg protein
compared to 2.36 ± 0.45 in protein lysates derived from control
muscle (Fig. 8C). Assuming that the underlying level of
endogenous mouse receptor protein expression is not reduced in
transgenics, the above results suggest that the majority of mouse
insulin receptors present in transgenic muscle had formed hybrid
complexes with mutant human receptors.
Figure 8:
Assessment of potential hybrid mouse-human
insulin receptors. Solubilized muscle (gluteal, 200 µg) proteins
derived from control or transgenic mice were subjected to quantitative
immunoprecipitation with a human-specific anti-insulin receptor
antibody (83-14). Immunoprecipitated proteins present in the
pellet (A) and remaining proteins present in the supernatant
(B) were resolved by SDS-PAGE followed by transfer to
nitrocellulose membranes and immunoblotting with a nonspecies-specific
anti-insulin receptor antibody. C, quantitation of mouse
insulin receptors present in solubilized gluteal muscle proteins
(supernatant) after depletion of human insulin receptors by
quantitative immunoprecipitation. Insulin receptors were immobilized on
microtiter wells coated with a nonspecies-specific anti-insulin
receptor antibody (AB-3) followed by incubation with
I-insulin, washing, and measurement of bound insulin. The
results represent mean ± S.E. of data from 4 control and 8
transgenic mice. Insulin binding was expressed as pmole of
-I-insulin bound/mg protein after subtracting values
corresponding to nonspecific binding
(<5%).
A similar approach was taken
to assess whether mouse IGF-1 receptor-human insulin receptor hybrids
were present in transgenic muscle. Following quantitative
immunoprecipitation of transgenic muscle lysates with 83-14, we
used an anti-IGF-1 receptor antibody (IGFR) to determine by
immunoblotting that a small amount of IGF-1 receptor
-subunit (a
100 kDa band) could be immunoprecipitated with human insulin receptor
protein (not shown). In contrast, no IGF-1 receptor protein was
detectable in 83-14 immunoprecipitates from control mouse muscle.
However, in comparison with the total amount of IGF-1 receptor present
in control or transgenic muscle, less than 10% of IGF-1 receptor
protein present in transgenic mouse muscle could be immunoprecipitated
with 83-14. These results suggest that there was only a small
degree of hybrid formation between murine IGF-1 receptors and mutant
human insulin receptors in transgenic mouse muscle.
insulin receptors expressed in CHO cells had severely impaired
insulin-stimulated phosphotransferase activity and failed to mediate
several biological responses to insulin
(4) . Subsequently, we
reported that overexpression of these mutant insulin receptors in the
muscle of transgenic mice was associated with a state of mild in
vivo insulin resistance and impaired muscle insulin receptor
tyrosine kinase activity
(22) . In the current study, we used
gluteal and gastrocnemius muscles from these mice where high levels of
the kinase-deficient dominant-negative receptors were expressed.
Importantly, total insulin-stimulated insulin receptor
autophosphorylation and phosphotransferase activity toward poly-Glu-Tyr
was severely reduced in both transgenic gluteal and gastrocnemius
muscles. This occurred with the use of a maximally stimulating insulin
dose as assessed by dose-response curves generated using control mouse
muscle. Thus, overexpression of mutant receptors exerted a
transdominant effect to abrogate insulin stimulation of endogenous
insulin receptors via impaired autophosphorylation and activation of
the tyrosine kinase.
-subunits to undergo
trans-phosphorylation mediated by residual wild type
-subunits
(36-38). Reduced muscle insulin receptor autophosphorylation in
our mouse model is consistent with the finding that insulin receptors
derived from a heterozygous patient with the Thr
receptor allele (where the ratio of mutant/normal receptor
protein is presumed to be 1:1) exhibited a 50% decrease in maximal
insulin stimulation of both receptor autophosphorylation and tyrosine
kinase activity
(39, 40) . Although total
insulin-stimulated phosphorylation of an exogenous substrate (histone)
was not impaired in fibroblasts overexpressing A/K 1018
receptors
(35) , expression of these same mutant receptors in
3T3-L1 cells
(36) did inhibit net insulin-stimulated
poly-Glu-Tyr phosphorylation in agreement with our results using muscle
from transgenic mice and the same insulin receptor kinase substrate.
/G
transition) and
differentiation
(45) . One aspect of these effects involves
stimulated expression of the immediate early gene, c-fos,
which is also regulated by insulin
(46, 47) . MAP kinase
may participate in c-fos gene induction via phosphorylation of
p62
- or via p90
-mediated phosphorylation of
the serum response factor since these transacting factors form a
complex that activates the c-fos promoter
(45) . Recent
reports showed that in vivo insulin administration to rats
stimulates p42 and p44 MAP kinase activation
(12, 48) in
skeletal muscle. Thus, insulin or IGF-1-mediated muscle differentiation
and development may involve the activation of MAP kinases.
heterodimers
composed of one mutant
half-receptor and one wild type
half-receptor are functionally inactive after assembly
in vitro(37) . Thus, hybrid formation has been invoked
as a potential mechanism for the transdominant effects of
kinase-deficient insulin receptors. Previous reports have also
suggested that transfected mutant human insulin receptors do form
hybrids with endogenous insulin receptors in cultured rodent
cells
(36, 56) , although other investigators have failed
to detect hybrid formation when mutant and (truncated) wild type
receptors were co-transfected
(38) .
300,000/cell) versus endogenous IGF-1 receptors
(
10,000/cell) favors hybrid formation and may thus contribute to
dominant-negative impairment of IGF-1 action in cultured cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.