From the
Insulin administration activates the insulin receptor kinase
(IRK) in both plasma membrane (PM) and endosomes (ENs) raising the
possibility of transmembrane signaling occurring in the endosomal
compartment. Peroxovanadium compounds activate the IRK by inhibiting
IR-associated phosphotyrosine phosphatase(s). Following the
administration of the phosphotyrosine phosphatase inhibitor
bisperoxo(1,10-phenanthroline)oxovanadate (V) anion (bpV(phen))
activation of the hepatic IRK in ENs preceded that in PM by 5 min. When
colchicine treatment preceded bpV(phen) administration IRK activation
in ENs was unaffected but was totally abrogated in PM. Insulin receptor
substrate-1 tyrosine phosphorylation followed the kinetics of IRK
activation in ENs not PM and a hypoglycemic response similar to that
achieved with a pharmacological dose of insulin ensued. These studies
demonstrate that ENs constitute a site for IR-mediated signal
transduction.
The insulin receptor (IR)
In 1992 Faure et al.(18) reported
the existence of phosphotyrosine phosphatase(s) (PTPs) in the ENs which
dephosphorylate the activated autophosphorylated IR leading to
attenuation of the insulin signal
(18) . More recently we have
described the insulin-mimetic properties of 12 crystallizable, >95%
pure, peroxovanadium compounds (pVs) each containing one or two peroxo
anions, an oxo anion, and an ancillary ligand (which confers stability
to the complex)
(19) . When administered in vivo these
compounds resulted in hypoglycemia and activation of hepatic IRK
(19, 20) . In contrast, activation of skeletal muscle
glycogen synthesis and IRK was induced by only selected compounds
(20) .
In the present study we have explored the activity of
the pV compound, bisperoxo(1,10-phenanthroline)oxovanadate(v) anion
(bpV(phen)). The in vivo administration of bpV(phen) results
in inhibition of endosomal IR-specific PTP(s) and corresponding
augmentation of endosomal IRK activity. The activation of endosomal IRK
preceded and exceeded that attained in PM. Pretreatment with colchicine
resulted in selective activation of the endosomal IRK by bpV(phen)
accompanied by tyrosine phosphorylation of insulin receptor substrate-1
(IRS-1) and hypoglycemia indicating an important role for the ENs in
regulating insulin signal transduction.
Earlier studies found that the administration of insulin is
rapidly followed by the accumulation of both insulin
(33) and
IRs
(26) in rat liver ENs. It was subsequently shown that these
internalized receptors were activated IRKs
(7) whose
autophosphorylation activity exceeded the maximal levels attained by PM
IRKs
(7) . These observations on the IRK and comparable studies
on the epidermal growth factor receptor kinase
(34, 35, 36) support a role for internalized receptors in transmembrane
signaling
(37) . In this paper we have described for the first
time an in vivo model in which there is selective activation
of the hepatic endosomal IRK following which there is tyrosine
phosphorylation of IRS-1 and hypoglycemia.
The data of
Fig. 1
suggest that bpV(phen) acts following entry into the cell.
Fig. 2
shows that ENs contain an IRK-associated PTP(s) whose
inhibition by bpV(phen) followed by insulin administration leads to
pronounced activation of the endosomal IRK. The striking correlation
between IRK activation on one hand and PTP(s) inhibition on the other
supports our earlier suggestion
(19) that these phenomena are
causally related. The relationship between PTP inhibition and IRK
activation deserves further comment as various studies have observed
that cultured cells display a low level of basal IRK activity even in
the absence of insulin
(9, 19) . It is envisioned that
in the basal state a low level futile cycle operates in which
phosphotyrosine is formed and degraded with no significant net
autophosphorylation. The administration of bpV(phen) disturbs this
equilibrium consequent to the inhibition of dephosphorylation leading
to net IRK autophosphorylation and hence activation. The time course of
IRK activation following bpV(phen) was slower than that following
insulin which was maximal at 2 min post-injection
(7, 8, 19) . This is consistent with the
different modes by which each agent promotes IRK activation. Thus
bpV(phen) activates the IRK indirectly by inhibiting IRK-associated
PTP(s), whereas insulin activates the IRK by augmenting kinase activity
directly.
To assess the primary site of action of bpV(phen) we
evaluated the kinetics of PM and EN IRK activation following bpV(phen)
administration. As seen in Fig. 3, bpV(phen) administration
produced a more rapid and greater activation of the IRK in ENs compared
to those of PM. This prompted the suggestion that the lag in and lower
level of PM IRK activation reflected a recycling of IRKs primarily
activated in ENs. We therefore attempted to abrogate receptor recycling
by pretreating the animals with the colchicine, a well known inhibitor
of microtubule function. Indeed colchicine treatment completely
prevented the increase in PM IRK activity following bpV(phen)
administration without affecting the extent of endosomal IRK
activation. One hour of colchicine treatment did not activate the IRK
in PM or ENs as measured by either the exogenous kinase assay or IRK
tyrosine phosphorylation ( cf. zero time in Figs. 3 and 4). Nor
did colchicine pretreatment alter the effect of insulin on the extent
of IRK activation in either PM or ENs (see ``Results'').
Therefore the most likely explanation for the selective loss of
bpV(phen)-induced IRK activation in the PM after colchicine
administration is the inhibition of exocytic transport of the activated
endosomal IRK to the PM.
These findings defined an in vivo system in which endosomal IRK could be selectively activated
following colchicine and bpV(phen) treatment, thus allowing us to
assess the possibility of signaling from this compartment alone. IRS-1,
an important substrate of the IRK, has been implicated in aspects of
the insulin signaling pathway
(38, 39) . Our study has
shown that IRS-1 became highly tyrosine phosphorylated by 5 min
following bpV(phen) treatment alone and by 15 and 30 min following
colchicine pretreatment, instances when the endosomal but not PM IRK
was active. This implies that the endosomal IRK was capable of normal
tyrosine phosphorylation of IRS-1 and hence of signaling. The level of
IRS-1 tyrosine phosphorylation was comparable to that seen with in
vivo administered insulin (1.5 µg/100 g body
weight).
Earlier work has
suggested that muscle accounts for 50%
(24) , liver for 30%
(42) , and adipose tissue for <10%
(24) of the uptake
of administered glucose. Several observations have indicated that the
in vivo hypoglycemic action of bpV(phen) derives primarily
from action on the liver rather than on skeletal muscle. Thus bpV(phen)
could not stimulate the incorporation of glucose into glycogen
(20) or the uptake of 2-deoxyglucose (Fig. 6 A)
into skeletal muscles. Thus our observation that bpV(phen)
administration would still lower blood glucose levels in
colchicine-pretreated rats (Fig. 6 B) implicates the
endosomal IRK of rat liver in effecting reduced hepatic glucose output.
The hypothesis that internalization of receptors may be involved in
transmembrane signaling
(37, 43) has been supported by
observations on the accumulation of activated IRKs
(7, 8) and epidermal growth factor receptor kinases
(34, 36) in ENs. This hypothesis has been strengthened by the recent
demonstration that accumulation of activated epidermal growth factor
receptor kinases in ENs leads to the phosphorylation of an in vivo substrate, pyp55
(44) (also called p55
(45) and
more recently identified as SHC
(46, 47) ), leading to
the association of SHC and GRB2 with the activated internalized
epidermal growth factor receptor kinase
(46) . The current study
has shown that selective activation of the hepatic endosomal IRK leads
to phosphorylation and recruitment of IRS-1 to the cytosol and the
lowering of blood glucose levels. It thus provides support for a role
for the EN in insulin signal transduction and illustrates the
importance of the intracellular IRK for the realization of some of the
metabolic effects of insulin.
Rats were fasted overnight and sacrificed 15
min after the injection of 0.6 µmol/100 g body weight of bpV(phen).
Hepatic PM and ENs were prepared, solubilized, and IRs partially
purified by lectin chromatography as described under
``Experimental Procedures.'' Immunoprecipitations were
performed in parallel with
Rats were fasted overnight and injected
intrajugularly with 0.6 µmol/100 g body weight bpV(phen) 60 min
after receiving an intrajugular injection of colchicine (25
µmol/100 g body weight) or vehicle. At the specified time animals
were sacrificed and PM and ENs prepared.
Rats were fasted overnight and
injected intrajugularly with 0.6 µmol/100 g body weight bpV(phen)
with or without colchicine pretreatment (25 µmol/100 g body
weight). At the specified time animals were sacrificed and cytosol
prepared. Tyrosine kinase activity (TKA) was assayed as described under
``Experimental Procedures.'' All data are the mean ±
S.E. from the noted number ( n) of separate animals.
We express our gratitude to Dr. Morris White for
generously providing the antibodies to IRS-1 used in these studies. We
also thank Gerry Baquiran and Qingwei Chu for their excellent technical
assistance and Dr. Jesse B. Ng for preparing the pV compound used in
these studies.
Addendum-Subsequent to the submission of this manuscript,
Kublaoui et al.(48) demonstrated that in rat
adipocytes IRS-1 was distributed between internal membranes (20%) and
cytosol (80%) with none detectable in the plasma membrane. Following
insulin treatment, tyrosine phosphorylation of IRS-1 paralleled that of
the IRK in internal membranes whereas no tyrosine-phosphorylated IRS-1
was observed in plasma membranes. This observation is consistent with
insulin signal transduction occurring within the endosomal compartment.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is a
heterodimeric glycoprotein
(1) consisting of two
-subunits
containing insulin binding sites
(2, 3) and two
transmembrane
-subunits possessing tyrosine kinase activity in
their cytosolic domains
(4, 5) . Insulin binding leads
to rapid tyrosine kinase activation, culminating in autophosphorylation
of the
-subunits
(6) and internalization of the activated
insulin-receptor complex into endosomes (ENs)
(7, 8) .
The accumulation of activated IRs in ENs is compatible with tyrosine
phosphorylation of proteins in the insulin signal transduction cascade
at locations topologically distinct from the PM
(7, 8) .
Studies utilizing insulin-mimetic agents
(9, 10) ,
kinase inhibitory antibodies
(11, 12) , and kinase
impaired receptor mutants
(13, 14, 15, 16) have defined the key role activation of the insulin
receptor kinase (IRK) plays in realizing the biological effects of
insulin
(17) . Taken together these considerations support the
notion that the internalized IRs are involved in insulin signal
transduction.
Animals
Female Sprague-Dawley rats, 10 weeks of
age (160-180 g body weight) were purchased from Charles River
Canada Ltd. (St. Constant, PQ). Except where indicated animals were
fasted for 16-18 h prior to experimentation.(
)
Materials
Porcine insulin was a gift
from Eli Lilly and Co., (Indianapolis, IN). Phenylmethanesulfonic
fluoride (PMSF), aprotinin, leupeptin, pepstatin A, HEPES (free acid),
sodium orthovanadate, rabbit -globulin, bacitracin, TRIS,
polyglutamic acid-tyrosine (4:1) (Glu:Tyr),
N-acetyl-D-glucosamine, radioimmunoassay grade bovine
serum albumin (BSA), colchicine, fetal and newborn calf serum,
Swim's 77 media, and most other chemicals were purchased from
Sigma. Wheat germ agglutinin-Sepharose 6MB (WGA-Sepharose) and Protein
A-Sepharose CL-4B were from Pharmacia LKB Biotechnology (Uppsala,
Sweden). Carrier-free Na[
I] and
-labeled
[
P]ATP (3000Ci/mmol) were from DuPont NEN
Radiochemicals (Lachine, PQ, Canada). Adenosine 5`-triphosphate,
disodium salt (ATP) was from Boehringer Mannheim (Laval, PQ, Canada).
2-Deoxy-D-[1-
H]glucose
(2-[
H]DG; 10.6 Ci/mmol) was from Amersham
International plc (Buckinghamshire, United Kingdom). Reagents for
electrophoresis were from Bio-Rad with the exception of
C-labeled protein standards which were supplied by Life
Technologies, Inc./BRL Canada (Burlington, Ontario, Canada).
Dulbecco's modified Eagle's medium and Ham's F-12
media were from Life Technologies, Inc./BRL. Penicillin, streptomycin,
and fungizone were purchased from Flow Laboratories, Inc. (McLean, VA).
Kodak X-Omat AR film was from Picker International (Montreal, PQ,
Canada). Immobilon-P transfer membranes were from Millipore Ltd.
(Mississauga, Ontario, Canada).
Preparation of Peroxovanadium Compound
bpV(phen)
The compound bpV(phen) was prepared by Dr. Jesse Ng of
the Department of Chemistry, McGill University as described elsewhere
(19, 21) .
Hepatoma Cell Cultures
H4IIEC3 cells were a gift
from Dr. D. K. Granner, Vanderbilt University. The cells were grown to
confluency in Corning 150-cm flasks in Swim's 77
media supplemented with 6 mM NaHC0
, 2.4
mM CaCl
, 1.2 mg/ml glucose, 60 mM
Tricine, 60 µM cysteine, 2 mM glutamine, 100
IU/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml fungizone,
10 µg/ml phenol red, and 2.5% (v/v) each of fetal and newborn calf
sera. The cells were placed in serum-free Dulbecco's modified
Eagle's medium/Hams's F-12 for 16-20 h prior to
treatment with reagents. One hour before treatment the medium was
replaced with 10 ml of Kreb's Ringer bicarbonate containing 1.0%
BSA. A 5-ml volume of a three times concentrated solution of bpV(phen)
and/or sodium orthovanadate was then added as indicated in the text.
Incubation at 37 °C was carried out for 20 min. Following the
incubation, cells were washed once with ice-cold phosphate-buffered
saline (PBS) and quick-frozen in ethanol-dry ice.
Solubilization and Lectin-affinity Purification of
Hepatoma Cells
To partially purify cellular IRs, cells from one
150-cm flask were suspended in 24 ml of 50 mM
HEPES, pH 7.6, 1 mM PMSF, 1% Triton X-100, 1 mM
ammonium molybdate, 2 µg/ml benzamidine, 0.3 trypsin inhibitory
units/ml aprotinin and shaken for 30 min at 4 °C. Insoluble
material was removed by centrifugation at 200,000
g
for 30 min and the supernatant was recycled
five times through 1-ml WGA-Sepharose columns which had been
equilibrated with 50 ml of 50 mM HEPES, pH 7.6, 0.1% Triton
X-100, 1 mM PMSF, 1 mM ammonium molybdate, 2
µg/ml benzamidine, 150 mM NaCl. Each column was washed
with 50 ml of 50 mM HEPES, pH 6.0, 10 mM
MgSO
, 0.1% Triton X-100, 1 mM PMSF, 1 mM
ammonium molybdate, 2 µg/ml benzamidine, 150 mM NaCl
followed by 20 ml of the pH 7.6 buffer. IRs were eluted from the
columns with 1.2 ml of pH 7.6 buffer containing 0.3 M N-acetyl-D-glucosamine and 0.3 trypsin inhibitory
units/ml aprotinin.
Antibodies
An antibody raised against a peptide
corresponding to the juxtamembrane region of the IR -subunit
(
960) was prepared and purified on a Protein A-Sepharose CL-4B
column as described previously
(8) . A polyclonal
IRS-1
antibody for immunoprecipitation and a monoclonal IRS-1 antibody for
Western blotting (1M92-7) were kindly provided by Dr. M. F. White
(Joslin Diabetes Center, Boston). A polyclonal phosphotyrosine antibody
(
PY) for immunoprecipitation was purchased from Upstate
Biotechnology Inc. (Lake Placid, NY). A monoclonal
PY for Western
blotting (P3300) was purchased from Sigma.
Injections
Following ether anesthesia rats
received an intrajugular injection of either bpV(phen) in PBS or
insulin in PBS containing 0.1% BSA for the times indicated. In some
instances animals received an injection of colchicine in 0.9% NaCl via
the same route.
Preparation of Plasma Membranes
Following
intrajugular injection, animals were sacrificed by decapitation at the
indicated times post-injection. The livers were exsanguinated and
rapidly excised prior to mincing at scissor point in ice-cold 5
mM TRIS-HCl buffer, pH 7.4, containing 0.25 M
sucrose, 1 mM benzamidine, 1 mM PMSF, 1 mM
MgCl, 2 mM NaF, and 2 mM sodium
orthovanadate. All preparative procedures were performed at 4 °C in
the presence of the same concentration of phosphatase/protease
inhibitors and buffer with only the sucrose concentration changing as
indicated. The livers were homogenized (4 ml of 0.25 M sucrose
buffer/g liver) with 10 passes of a loose fitting B pestle in a 50-ml
Wheaton homogenizer. The homogenate was centrifuged at 225
g
for 6 min in a JA-17 Beckman rotor to produce a
supernatant (S1) and pellet (P1). S1 was retained and the P1
resuspended with three passes of the B pestle in half the original
volume of 0.25 M sucrose buffer and centrifuged as before to
yield P2, which was discarded, and S2 which was pooled with S1. The
combined S1 and S2 were then centrifuged at 1,600
g
for 10 min to yield S3 and P3. P3 was retained,
resuspended in a final volume of 40 ml, and adjusted to a final sucrose
concentration of 1.42 M. This was overlaid with 3 ml of 0.25
M sucrose buffer and centrifuged at 83,000
g
for 1 h. The interface was removed, adjusted to
a final sucrose concentration of 0.39 M and recentrifuged at
1,600
g
for 10 min as before. The pellet
which constituted the PM was resuspended in 0.25 M sucrose
buffer.
Preparation of Endosomes
Following intrajugular
injection, animals were sacrificed by decapitation at the indicated
time post-injection. The livers were exsanguinated and rapidly excised
prior to mincing in 0.25 M sucrose buffer. The livers were
homogenized (4 ml of 0.25 M sucrose buffer/g liver) in a
Potter-Elvehjem homogenizer with 6 passes of a motorized Teflon pestle
at 1,500 rpm. The homogenate was centrifuged at 3,300
g
for 10 min in a Beckman Ti 50.2 fixed angle
rotor to yield S1 and P1. S1 was then centrifuged at 200,000
g
for 40 min in the 50.2 Ti rotor to yield S2
which was discarded and P2, the microsomal fraction. This was
resuspended and adjusted to a final sucrose concentration of 1.15
M and placed below 1.0 and 0.6 M sucrose solutions.
Following centrifugation at 96,500
g
for
205 min in a Beckman SW 28 rotor, the EN fraction at the 0.6/1.0
M interface was removed. ENs were then diluted in 0.25
M sucrose buffer and pelleted by centrifugation at 200,000
g
for 40 min in the 50.2 Ti rotor. The
pellet was resuspended in 0.25 M sucrose buffer.
Preparation of Cytosol
A 20% liver homogenate was
prepared as described for the EN preparation and the supernatant,
constituting the cytosolic fraction, was separated following
centrifugation at 200,000 g
for 45 min in
a SW40 Beckman rotor.
Solubilization and Lectin-affinity Purification of Plasma
Membrane and Endosomal Insulin Receptors
PM and ENs were
suspended in 4.5 ml of 0.25 M sucrose buffer as described
above but additionally containing 20 µM leupeptin, 20
µM pepstatin A, and 0.3 trypsin inhibitory units/ml
aprotinin. A 0.9-ml fraction was kept for analysis and the remaining
3.6 ml was solubilized by the addition of 0.4 ml of 0.25 M
sucrose buffer containing 10% Triton X-100. The fractions were
solubilized for 1 h at 4 °C on an Eberbach shaker (Ann Arbor, MI)
at low speed followed by centrifugation at 200,000
g
for 30 min to remove insoluble material. The
solubilized material was recycled five times over 2-ml WGA-Sepharose
columns which had been pre-equilibrated with 80 ml of 50 mM
HEPES buffer, pH 7.6, containing 150 mM NaCl, 1 mM
benzamidine, 1 mM PMSF, 0.1% Triton X-100, and 2 mM
sodium orthovanadate. The columns were then washed with 80 ml of 50
mM HEPES buffer, pH 6.0, containing 150 mM NaCl, 10
mM MgSO
, 1 mM benzamidine, 1 mM
PMSF, 0.1% Triton X-100, and 0.1 mM sodium orthovanadate to
remove any insulin still bound to the receptor, followed by 20 ml of
the above buffer adjusted to pH 7.6, to return the column to
physiological pH. The receptor was eluted with 2 ml of the 50
mM HEPES buffer, pH 7.6, containing 0.3 M N-acetyl-D-glucosamine (elution buffer).
Insulin Binding
Determination
I-Insulin was prepared using the
chloramine-T method as described previously
(22) and had a
specific activity of 130-200 µCi/µg. Specific binding of
I-insulin to intact and lectin-purified receptor was
determined at three different concentrations after overnight incubation
at 4 °C as described previously
(8) .
Protein Assay
Protein content was determined by a
modification of Bradford's method using BSA as standard
(23) .
Measurement of IRK Activity
Exogenous tyrosine
kinase activity was determined using
poly(Glu:Tyr
) as the substrate. Each assay was
performed with an amount of IR which bound 10 fmol of
I-insulin in the binding assay as described previously
(8) .
Immunoprecipitation of WGA-purified
Fractions
WGA-Sepharose purified IR (100 fmol) was diluted to a
final volume of 250 µl in elution buffer. Fifty microliters of
control IgG, PY, or
960 were added and incubated at 4 °C
for 4 h on a shaker at low speed. Next 150 µl of a 50% slurry of
Protein A-Sepharose CL-4B was added and the mixture shaken for a
further 1 h. The Protein A-Sepharose CL-4B was then pelleted by
centrifugation at 12,000
g
in an
Eppendorf microcentrifuge for 5 min at 4 °C. The supernatant was
carefully removed and assayed for exogenous tyrosine kinase activity
and insulin binding.
Immunoprecipitation of IRS-1
Cytosolic protein (16
mg) was incubated in the presence of Triton X-100 (1% final
concentration) for 1 h at 4 °C in a volume of 1.1 ml. Any insoluble
material was removed by centrifugation at 12,000
g
for 5 min in an Eppendorf microcentrifuge
following which 10 µg of
IRS-1 in 10 µl was added and the
incubation continued for a further 4 h. A 50% slurry of protein-A
Sepharose (150 µl) pre-equilibrated in 50 mM HEPES, pH
7.4, 150 mM NaCl, 2 mM sodium orthovanadate, was
added, shaken for a further 1 h, centrifuged as before and the pellet
rinsed with 1 ml of wash buffer (50 mM HEPES, pH 7.4;
containing 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 100
mM NaF, and 2 mM sodium orthovanadate) three times
followed by boiling in 210 µl of Lammeli sample buffer (2.3% SDS,
10% glycerol, 100 mM dithiothreitol, and 0.37 M
Tris-HCl, pH 6.8: final concentration) for 5 min. Seventy-microliter
samples were subjected to SDS-PAGE (7.5% gel) and then transferred to
Immobilon-P membranes. PY and IRS-1 content of the immunoprecipitates
were determined by Western blotting using P3300 and 1M92-7
antibodies, respectively, visualized by autoradiography, and quantified
by laser densitometry.
Western Blotting
For analysis of PY, IR, or IRS-1
content, either equal amounts of protein or femtomole of IR binding
capacity were used and 3 Lammeli sample buffer (6.9% SDS, 30%
glycerol, 300 mM dithiothreitol, and 1.1 M Tris-HCl,
pH 6.8) was added. The samples were boiled for 5 min and subjected to
SDS-PAGE under reducing conditions before electrophoretic transfer of
phosphoproteins from SDS gels to nitrocellulose membranes
(8) .
After transfer the membranes were blocked for 1 h (see below). The
blocking solution was then exchanged for a 50-ml solution containing
the primary antibody and gently shaken for 2 h at room temperature.
This was followed by three 10-min washes with 50 ml of washing solution
before the blots were then transferred to 50 ml of
I-labeled secondary antibody solution (700,000
cpm/electrophoretic lane transferred) for 1 h at room temperature,
followed by three additional washes. The nitrocellulose membranes were
mounted on Whatman 3MM paper squares and allowed to air dry. Labeled
proteins were visualized by autoradiography at -70 °C using
enhancing screens and Kodak X-Omat AR film. For PY Western blots all
incubations were in the presence of PBS containing 20% fetal calf serum
and washes were in PBS containing 1% Tween 20. For IR Western blots all
incubations were in the presence of PBS containing 4% powdered milk and
washes were in PBS containing 1% Tween 20. For IRS-1 Western blots all
procedures were in the presence of 10 mM TRIS, pH 7.4, 300
mM NaCl, and 0.05% Tween 20 (TNT) containing 3% BSA and washes
were in TNT.
IR Dephosphorylation Assay
IR dephosphorylation
was assessed in rat liver ENs prepared 2 min following the intrajugular
injection of 1.5 µg of insulin/100 g of body weight as described
previously
(18) . Briefly, ENs (25 µg) were incubated at 37
°C with 25 µM [-
P]ATP for
5 min. EDTA and unlabeled ATP at final concentrations of 10 and 500
µM, respectively, were then added to terminate
P labeling of the IR and allow dephosphorylation to occur.
After 10 min of further incubation at 37 °C, ENs were solubilized,
immunoprecipitated overnight at 4 °C with IR antibody, and
subjected to SDS-PAGE on a 7.5% gel. Phosphoserine and phosphothreonine
were removed by KOH digestion, the gels dried, and the phosphotyrosine
content of the IR visualized by autoradiography. The extent of
[
P] loss from the IR
-subunit, measured in
the presence and absence of bpV(phen), was used to calculate the
percent inhibition effected by bpV(phen).
Measurement of Plasma Glucose Levels
Rats were
fasted for 4 h prior to excision of the tail tip. Following a further
30 min (recovery period), 60-µl blood samples were obtained to
assess basal plasma glucose values. Animals were then injected
intravenously with either 0.9% saline or colchicine (25 µmol/100 g
body weight) via the tail vein. One hour later PBS or bpV(phen) was
injected and blood samples collected into heparinized tubes at 15-min
intervals thereafter. Plasma was obtained by centrifugation at 12,000
g
for 5 min (Beckman Microfuge B) and
plasma glucose concentrations determined in duplicate using a glucose
analyzer-2 (Beckman instruments) as previously noted
(19) . Uptake of
2-Deoxy[1-
H]glucose-Following the
intrajugular injection of a combination of 18.75 µCi/100 g body
weight 2-[
H]DG with either PBS, 0.6 µmol/100
g body weight bpV(phen) or 1.5 µg/100 g body weight insulin, the
animals were sacrificed at 15 min post-injection time by decapitation
and the bodies exsanguinated. Diaphragm, soleus, tibialis anterior and
extensor digitorum longus muscles were removed and muscle
2-[
H]DG-6-phosphate content determined as
described elsewhere
(24) .
Statistical Analysis
Welch's test was used
to analyze for significant differences between groups provided that the
samples were normally distributed. If the groups differed significantly
from a normal distribution then the Wilcoxon rank sum test was employed
(25) .
Effect of Vanadate on IRK Activation and Inhibition of
Endosomal PTPs by bpV(phen)
It was previously demonstrated that
incubation of H4IIEC3 hepatoma cells for 20 min with 1 mM
bpV(phen) produced activation of hepatoma cell IRK, whereas incubation
with 1 mM vanadate was virtually ineffective
(19) .
Fig. 1
demonstrates that the incubation of bpV(phen) (1
mM) with increasing concentrations of vanadate (0.01-10
mM) resulted in a dose-dependent inhibition of IRK activation.
In contrast, increasing concentrations of vanadate did not
significantly affect the inhibitory influence of bpV(phen) on endosomal
IRK dephosphorylation (Fig. 1). Together these observations
suggest that vanadate competes for a common cellular transport process
with bpV(phen) but is ineffective at a major site of bpV(phen) action,
the inhibition of IRK dephosphorylation. They further imply that
bpV(phen) action follows its entry into the cell.
Figure 1:
Effect of vanadate on the activation
of the IRK and inhibition of endosomal PTPs by bpV(phen). To assess the
effect of vanadate on the activation of the IRK by bpV(phen), H4IIEC3
hepatoma cells were incubated in the presence of 1 mM
bpV(phen) and the indicated concentration of vanadate for 20 min. The
cells were then washed, solubilized, IRs partially purified by lectin
chromatography, and the IRK activity measured as described under
``Experimental Procedures.'' Each point is the mean ±
S.E. of two to five separate determinations. To assess the effect of
vanadate on inhibition of endosomal IR tyrosine dephosphorylation,
animals were injected with 1.5 µg/100 g body weight insulin for 2
min, ENs were prepared, and the PTP assay was performed in the presence
of 10 µM bpV(phen) and the indicated concentrations of
vanadate as described under ``Experimental Procedures.'' Each
observation is the mean ± S.E. of two to five separate
determinations.
IRK Activation and PTP Inhibition in ENs following the in
Vivo Administration of bpV(phen) and Insulin
We previously
demonstrated the accumulation of maximally activated IRK within hepatic
ENs by 2 min following in vivo insulin administration
(7, 8) . More recently we showed that prior treatment of
rats with bpV(phen) greatly augmented insulin-induced endosomal IRK
activation and that there was a significant correlation between IRK
activation in hepatoma cells and the inhibition of IRK
dephosphorylation in hepatic ENs by pVs
(19) . In the present
study we sought to examine this correlation further by comparing IRK
activation and the inhibition of IRK dephosphorylation within the same
hepatic endosomal system. Rats were pretreated with bpV(phen) from 5
min to 6 h and, at each time, were sacrificed 2 min after insulin
injection (Fig. 2). The tyrosine kinase activity of IRKs,
partially purified from ENs by WGA chromatography, was assessed using
PGT as substrate. The inhibition of IRK dephosphorylation and hence of
IRK-associated PTP(s) activity was measured by assessing the loss of
[P] from autophosphorylated endosomal IRKs as
described under ``Experimental Procedures.''
Fig. 2A shows that the in vivo administration
of bpV(phen) anywhere between 5 min and 6 h before insulin
administration augmented insulin-stimulated IRK activation in a
time-dependent manner. Maximal activation was seen at 15-45 min
with a subsequent decrease of the activation state to approximately
baseline levels by 5 h after bpV(phen) injection. The time course and
extent of inhibition of IRK-associated PTP activity closely followed
that of IRK activation (Fig. 2 A). There was thus a
strong correlation between the in vivo activation of the
endosomal IRK and inhibition of the corresponding IRK-associated PTP(s)
(Fig. 2 B; r = 0.97, p <
0.001).
Figure 2:
Correlation between IRK activation and PTP
inhibition in ENs after treatment with bpV(phen) and insulin. Rats were
fasted overnight and given an intrajugular injection of 0.6
µmol/100 g body weight bpV(phen). They then received a second
injection of 1.5 µg/100 g body weight insulin 2 min prior to
sacrifice at the indicated times. Hepatic ENs were prepared and IRK and
PTP activities measured as described under ``Experimental
Procedures.'' Panel A, the time course of activation of
the IRK and the corresponding inhibition of PTP activity. Panel
B, the linear correlation between IRK activation and PTP
inhibition at each time point. Each observation is the mean of
determinations on two to five animals.
Time Course of IRK Activation and Tyrosine
Phosphorylation in PM and ENs after bpV(phen)
The above
observations suggest that bpV(phen) acts by entering the cell and
inhibiting IRK-associated PTP(s) resulting in IRK activation. We next
sought to evaluate the time course and extent of IRK activation in
vivo and focused on PM and ENs, the two major subcellular
compartments in rat liver harboring IRs
(7, 26) . Using
well-characterized methods to separate PM from ENs
(7, 8) we measured IRK activation in these cell fractions by
assessing IRK-mediated phosphorylation of PGT as well as the
phosphotyrosine content of the IRK. Following the in vivo administration of bpV(phen) activation of the IRK in ENs was more
rapid and significantly greater than that observed in PM
(Fig. 3 A). There was a reproducible lag of about 5 min
in IRK activation in PM, whereas no such lag was observed in ENs. In
addition, 87.7% ± 3.1% (S.E.; n = 3) of the
tyrosine kinase activity of lectin-purified endosomal IRK preparations
(15 min after bpV(phen)) was immunoprecipitated by 960, an IRK
specific antibody
(8) , confirming IRK as the major tyrosine
kinase activated by bpV(phen) treatment (). Furthermore,
the phosphotyrosine content of the endosomal IRK was much greater than
that in PM (Fig. 3 B) with a clear signal detectable for
the EN, but not PM IRK by 5 min. Treatment with bpV(phen) did not
produce a change in the IR content of either PM or ENs as assessed by
insulin binding () or immunoblotting with
960 (data
not shown). Thus, unlike the situation following insulin administration
(8) the administration of bpV(phen) was not followed by the
recruitment of IRKs into ENs from PM.
Figure 3:
Time course of activation and
phosphotyrosine content of the PM and EN IRK by bpV(phen). Rats were
fasted overnight and given an intrajugular injection of 0.6
µmol/100 g body weight bpV(phen). Following sacrifice at the times
indicated, hepatic PM and ENs were prepared, solubilized, and IRs
partially purified by lectin chromatography as described under
``Experimental Procedures.'' Panel A, the IR content
of PM () and ENs (
) was determined by measuring
I-insulin binding and exogenous tyrosine kinase activity
assessed using PGT as substrate as described under ``Experimental
Procedures'' and expressed as pmol/10 min/10 fmol of insulin
binding. Each point reflects the mean ± S.E. of determinations
on 3-13 separate animals. Panel B, 50 fmol of insulin
binding was loaded onto a 7.5% gel for SDS-PAGE. Proteins were
transferred onto PVDF Millipore Immobilon-P membranes and the PY
content of the IR
-subunit assessed by Western blotting using a PY
antibody (
PY) and a
I-labeled goat anti-mouse second
antibody as described under ``Experimental
Procedures.''
Time Course of bpV(phen)-induced IRK Activation in PM and
ENs following Colchicine Pretreatment
The delay in and lower
level of IRK activation in PM compared to ENs, along with the failure
of IRs to redistribute after bpV(phen) administration, suggested that
the primary site of IRK activation may be in ENs with changes in PM IRK
reflecting recycling of IRs from ENs back to PM. We elected to employ
colchicine, an inhibitor of microtubular function, to prevent receptor
recycling in liver parenchyma. This was based on the known microtubule
requirements for exocytosis in liver parenchyma and other mammalian
cells
(27, 28, 29) and the microtubule
requirement for endosomal transport to the Golgi-bile canalicular
region for late endosomal/lysosomal maturation in liver parenchyma
(30, 31, 32) . Recently we have observed that
colchicine inhibits recycling of the hepatic prolactin
receptor.(
)
We thus administered colchicine 1 h
prior to bpV(phen) treatment to determine whether selective activation
of endosomal IRKs occurred in this circumstance. Colchicine treatment
60 min prior to bpV(phen) administration resulted in IRK activation
exclusively in ENs with the level attained being identical to that in
rats not receiving colchicine pretreatment (Fig. 4 A). In
this circumstance IRK tyrosine phosphorylation was seen only in ENs
(Fig. 4 B) and there was little redistribution of IR
content as compared to that seen with bpV(phen) alone ().
As a further control we assessed the effect of colchicine treatment on
insulin-stimulated activation of the hepatic IRK. Colchicine was
administered to rats 60 min prior to insulin injection and IRK activity
was assessed at 30 s and 2 min post-insulin in PM and ENs,
respectively, the previously established times of maximal IRK
activation in these compartments
(8) . Following insulin
administration, IRK activity (pmol/10 min/10fmol insulin binding; mean
± S.D., n = 3) in PM was 19.3 ± 3.1
without and 18.1 ± 1.5 with colchicine pretreatment ( p = 0.58); while that in ENs was 14.3 ± 2.7 without
and 18.5 ± 2.7 with colchicine pretreatment ( p =
0.13). Hence colchicine did not impair IRK activation in either PM or
ENs, and we thus ascribe its effect to its activity as an inhibitor of
membrane recycling.
Figure 4:
Time course of activation of the PM and EN
IRK by bpV(phen) following colchicine treatment. Rats were fasted
overnight and given an intrajugular injection of 25 µmol/100 g body
weight colchicine in 0.9% saline 1 h prior to injection with 0.6
µmol/100 g body weight bpV(phen) and sacrificed at the indicated
times. Hepatic PM and ENs were prepared, solubilized, and IRs partially
purified by lectin chromatography as described under
``Experimental Procedures.'' Panel A, the IR content
of PM () and ENs (
) was determined by measuring
I-insulin binding and exogenous tyrosine kinase activity
assessed using PGT as substrate as described under ``Experimental
Procedures'' and expressed as pmol/10 min/10 fmol of insulin
binding. Each point reflects the mean ± S.E. of determinations
on three to four separate animals. Panel B, 20 fmol of insulin
binding was loaded onto a 7.5% gel for SDS-PAGE. Proteins were
transferred onto PVDF Millipore Immobilon-P membranes and the PY
content of the IR
-subunit assessed by Western blotting using a PY
antibody (
PY) and a
I-labeled goat anti-mouse second
antibody as described under ``Experimental
Procedures.''
Phosphotyrosine Content of IRS-1 following bpV(phen)
Treatment in the Presence and Absence of Colchicine
Using a
combination of colchicine and bpV(phen) we have generated an in
vivo system wherein only hepatic endosomal IRK was activated. In
order to determine whether insulin signaling could arise from this
compartment we assessed whether tyrosine phosphorylation of IRS-1 could
be observed in this situation. As can be seen in Fig. 5the
administration of bpV(phen) after colchicine augmented PY
phosphorylation of IRS-1 in a time-dependent fashion above that
observed in control animals (3-fold at 15 min ( p < 0.02)
and 5-fold at 30 min ( p < 0.01)). Even in the absence of
colchicine pretreatment, increased IRS-1 phosphorylation was observed 5
min after bpV(phen) when PM IRK activation was not evident
(Fig. 3 A). Also of interest is the observation that
bpV(phen) treatment resulted in an increase in the amount of IRS-1
present in the cytosol compared to that observed in controls (2-fold at
15 min ( p < 0.05) and 3-fold at 30 min ( p <
0.01)). The possibility that IRS-1 phosphorylation had occurred via a
cytosolic tyrosine kinase activated by bpV(phen) seems unlikely since
cytosolic tyrosine kinase activities were not augmented either in the
absence or presence of colchicine treatment (I).
Additionally in hepatoma tissue culture cells overexpressing kinase
negative IRK (K1030A mutation) versus those overexpressing
normal IRK, treatment with bpV(phen) resulted in no phosphorylation of
IRS-1 in the former but IRK activation and IRS-1 phosphorylation in the
latter.(
)
Effect of bpV(phen) on Plasma Glucose Levels and
2-[
H]DG Uptake in Rat Diaphragm-We
have previously shown that bpV(phen) administration alone causes
hypoglycemia in rats
(19) . Based on the inability of bpV(phen)
to stimulate glycogen synthesis in muscle
(20) we have
suggested that the locus of bpV(phen) action is the liver and not
muscle for producing the hypoglycemic response. An hepatic locus of
bpV(phen) action was further supported by the observation that
bpV(phen) did not stimulate 2-deoxyglucose uptake into rat skeletal
muscle in contrast to the significant stimulation induced by insulin
(Fig. 6 A). It is of interest that following the
selective activation of the endosomal IRK produced by bpV(phen) in
colchicine pretreated rats, hypoglycemia was induced
(Fig. 6 B) to a similar degree to that achieved with
bpV(phen) in the absence of colchicine pretreatment
(19) .
Figure 5:
Phosphotyrosine content of IRS-1 following
bpV(phen) treatment in the presence and absence of colchicine. Rats
were fasted overnight and given an intrajugular injection of 0.6
µmol/100 g body weight bpV(phen). In some cases rats were injected
intrajugularly with 25 µmol/100 g body weight colchicine in 0.9%
saline 1 h prior to bpV(phen) injection. A 20% liver homogenate was
prepared as described under ``Experimental Procedures'' and
the cytosolic fraction separated following centrifugation at 200,000
g
for 45 min. IRS-1 was
immunoprecipitated from the cytosol by
IRS-1, the pellet washed
three times, boiled in Lammeli sample buffer, and subjected to SDS-PAGE
(7.5% gel) as described under ``Experimental Procedures.'' PY
and IRS-1 content of the immunoprecipitates were determined by Western
blotting using specific antibodies, visualized by autoradiography, and
quantified by laser densitometry. Panel A, immunoblotting of
immunoprecipitated IRS-1 with antibodies to PY and IRS-1, respectively.
Panel B, the quantification of PY and IRS-1 immunoblots
following laser densitometry of the autoradiograph. *, p <
0.02; **, p < 0.01.
Figure 6:
Effect of bpV(phen) on 2-deoxyglucose
uptake into rat skeletal muscles and blood glucose levels in colchicine
pretreated rats. Panel A, uptake of
2-[H]DG into diaphragm ( DIA), soleus,
tibialis anterior ( TA), and extensor digitorum longus
( EDL) was assessed in overnight fasted rats. Animals were
sacrificed 15 min following intrajugular injection of a combination of
18.75 µCi/100 g body weight 2-[
H]DG with
either PBS, 0.6 µmol/100 g body weight bpV(phen) or 1.5 µg/100
g body weight insulin. Muscles were analyzed for their content of
2-[
H]DG-6-phosphate as described under
``Experimental Procedures.'' The content of
2-[
H]DG-6-phosphate in rat skeletal muscles:
DIA, soleus, TA, and EDL is shown following PBS ( open
bars), bpV(phen) ( slanted bars), or insulin ( solid
bars) treatment. Each observation is the mean ± S.E. of
determinations performed on three to six separate animals. *, p < 0.05. Panel B, rats were fasted for 4 h and then
injected with 25 µmol/100 g body weight colchicine in 0.9% saline
or 0.9% saline alone via the tail vein. One hour later rats received a
second injection of either PBS or bpV(phen) (0.6 µmol/100 g body
weight) via the same route. Tail blood samples were collected every 20
min for 3 h into heparinized Eppendorf tubes and centrifuged for 5 min
at 14,000
g
. Plasma glucose concentration
was determined in duplicate using a glucose analyzer-2 (Beckman
Instruments). The effect of bpV(phen) following colchicine treatment
(
) on plasma blood glucose levels is expressed as a percent of
plasma glucose levels following colchicine treatment alone (
).
Each point is the mean ± S.E. of determinations performed on
9-10 animals.
(
)
It was further seen that bpV(phen)
treatment caused recruitment of IRS-1 to the cytosol. This recruitment
to the cytosol suggests that in the nonphosphorylated state IRS-1 may
be associated with cell membrane fractions, including ENs
(40) ,
possibly via direct interactions with the IRK
(41) . The
recruitment to cytosol may be a necessary part of the IRK signaling
pathway allowing docking with other signal transducers and facilitating
their interactions with downstream elements in the signaling pathway
(38, 39) . However, it cannot be excluded that tyrosine
phosphorylation of IRS-1 renders it more immunoreactive which could
augment both the amount of IRS-1 immunoprecipitated and/or the strength
of the signal generated on immunoblotting.
Table:
Immunoprecipitation of lectin-purified PM and EN
receptors with 960
960 or control IgG on these partially
purified IRs. Subsequent supernatants were assayed for tyrosine kinase
activity (TKA) and
I-insulin binding (B) as described
under ``Experimental Procedures.'' The percentage of tyrosine
kinase activity and B immunoprecipitated was determined by subtracting
supernatant values for the immunoprecipitations with
960 from
those derived with control IgG. All values are the mean ± S.E.
of determinations on three to five separate animals.
Table:
Effect of bpV(phen) and colchicine on IR
content of PM and ENs
I-Insulin
binding was determined as described under ``Experimental
Procedures.'' All values are the mean ± S.E. from the noted
number (parentheses) of separate animals.
Table:
Effect of bpV(phen) and colchicine on
cytosolic tyrosine kinase activities
H]DG,
2-deoxy[1-
H]glucose; EN, endosome; IRK, insulin
receptor tyrosine kinase; IRS-1, insulin receptor substrate-1; PGT,
Glu:Tyr, polyglutamic acid-tyrosine (4:1); PM, plasma membrane; PMSF,
phenylmethanesulfonic fluoride; PTP, phosphotyrosine phosphatase; WGA,
wheat germ agglutinin; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
PY,
-phosphotyrosine; PAGE, polyacrylamide gel
electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.