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(Received for publication, July 8, 1997)
From the Department of Clinical Biochemistry, University of
Cambridge, Addenbrooke's Hospital, Hills Road,
Cambridge CB2 2QR, United Kingdom
Insulin signal transduction, initiated by binding
of insulin to its receptor at the plasma membrane, activates the
intrinsic receptor tyrosine kinase and leads to internalization of the
activated ligand-receptor complex into endosomes. This study addresses
the role played by the activated insulin receptor within hepatic
endosomes and provides evidence for its central role in
insulin-stimulated events in vivo. Rats were treated with
chloroquine, an acidotrophic agent that has been shown previously to
inhibit endosomal insulin degradation, and then with insulin. Livers
were removed and fractionated by density gradient centrifugation to
obtain endosomal and plasma membrane preparations. Chloroquine
treatment increased the amount of receptor-bound insulin in endosomes
at 2 min after insulin injection by 93% as determined by exclusion
from G-50 columns and by 90% as determined by polyethylene glycol
precipitation (p < 0.02). Chloroquine treatment also
increased the insulin receptor content of endosomes after insulin
injection (integrated over 0-45 min) by 31% when compared with
controls (p < 0.05). Similarly, chloroquine increased
both insulin receptor phosphotyrosine content and its exogenous
tyrosine kinase activity after insulin injection (64%;
p < 0.01 and 96% and p < 0.001, respectively). In vivo chloroquine treatment was without
any observable effect on insulin binding to plasma membrane insulin
receptors, nor did it augment insulin-stimulated receptor
autophosphorylation or kinase activity in the plasma membrane.
Concomitant with its effects on endosomal insulin receptors, chloroquine treatment augmented insulin-stimulated incorporation of
glucose into glycogen in diaphragm (p < 0.001). These
observations are consistent with the hypothesis that
chloroquine-dependent inhibition of endosomal
insulin receptor dissociation and subsequent degradation
prolongs the half-life of the active endosomal receptor and
potentiates insulin signaling from this compartment.
Insulin signal transduction is initiated by binding of insulin to
its receptor at the plasma membrane, which in turn leads to the rapid
autophosphorylation of multiple tyrosine residues on the intracellular
portion of the The anti-malarial drug chloroquine has been shown to elicit a number of
effects on insulin metabolism. Thus, in rats, chloroquine treatment
leads to hepatic retention of intact insulin within endosomes (12, 20)
due to inhibition of endosomal insulin degradation (13, 21, 22). This
effect does not rely solely on the acidotrophic nature of chloroquine
(whereby it is accumulated within acidic vesicles neutralizing the pH;
Ref. 23), as chloroquine's inhibition of insulin degradation persists
in detergent disrupted endosomes (13, 21). Kinetic analysis of the
rates of endosomal insulin degradation in the absence of chloroquine
show it to be a bi-exponential process where the values of the rate
constants are very similar to those for the dissociation of insulin
from its receptor (21), suggesting that dissociation from the receptor is the rate-limiting step in degradation. Chloroquine results in a
2-fold decrease in the rate constant of the slow process, together with
an increase in the proportion of degradation proceeding via the slow
process (21, 24). Additionally, chloroquine has been shown to increase
insulin binding in cultured hepatoma cells (25, 26) and IM9 lymphocytes
(27). Modeling of insulin binding to purified plasma membranes in the
presence of chloroquine demonstrated that the augmentation of binding
was due to a decrease in the rate constant for insulin dissociation
(28). Clinical manifestations of chloroquine action have also been
observed. Thus, in non-insulin-dependent diabetes mellitus,
chloroquine improves glucose tolerance (29), increases peripheral
glucose disposal, and decreases the metabolic clearance rate of insulin
(30). In insulin-dependent diabetes mellitus, chloroquine
has been shown to reduce insulin resistance (31).
This augmentation of insulin-receptor interaction observed with
chloroquine treatment prompted the present study to ascertain whether
in vivo chloroquine treatment in the rat: 1) increases the
amount of insulin bound to endosomal insulin receptors, 2) inhibits
dissociation and hence degradation of insulin within endosomes, 3)
augments and/or prolongs the autophosphorylation state and activity of
the endosomal insulin receptor tyrosine kinase, 4) affects the temporal
flux of insulin receptors through the endosome compartment, and 5)
augments insulin-stimulated metabolic events.
Male Sprague-Dawley
rats1 (180-200 g, body
weight) were fed ad libitum on a standard chow diet and
housed at 22 ± 1 °C with 12-h light cycles.
Porcine insulin suspension was purchased from
Calbiochem (Notts, UK). Chloroquine
(7-chloro-4-(4-diethylamino-1-methylbutylamino)quinoline), RIA2-grade BSA and most other
chemicals were purchased from Sigma (Poole, Dorset, UK). Carrier-free
Na125I and [ Monocomponent porcine insulin was
prepared from insulin zinc suspension (British Pharmaceuticals) as
outlined by Christensen et al. (33) and was iodinated with
lactoperoxidase (34). The 125I-[A14]insulin isomer was
separated by HPLC as described previously (35).
A monoclonal phosphotyrosine antibody (4G10)
used for immunocapture assays was purchased from Upstate Biotechnology
Inc. (Lake Placid, NY). A monoclonal antibody (CT-3) raised to, and
reacting with, the last 43 amino acids of the insulin receptor
Following Metafane inhalation anesthesia, rats
received an intraperitoneal injection of either 20 µmol/100 g body
weight chloroquine in PBS, pH 7.4, or PBS alone at 2 and 1 h prior
to insulin injection. Insulin (0.15 or 1.5 µg/100 g body weight) was
administered via intrajugular injection in PBS, pH 7.4, containing
0.1% RIA-grade BSA. Animals were sacrificed after insulin injection at
the times indicated in figure legends. In studies where PEG
precipitation, HPLC, and gel permeation chromatography were under
investigation, animals were anesthetized with an intraperitoneal
injection of Sagital (60 mg/kg body weight). Following laparotomy,
125I-[A14]insulin (1 MBq in 0.5 ml of PBS, 5% RIA-grade
BSA) was injected via the hepatic portal vein over a period of 30 s and the liver removed after another 2 min.
Following
sacrifice, livers were removed rapidly and placed in ice-cold
homogenization buffer. Plasma membranes were prepared as described by
Bevan et al. (10) and endosomes prepared as described below.
Livers were minced with scissors in ice-cold homogenization buffer (10 mM HEPES buffer, pH 7.4, containing 0.25 M
sucrose, 1 mM MgCl2, 0.1 mM
phenylmethanesulfonic fluoride, 0.2 mM AEBSF, 0.02 mM E-64, 0.02 mM pepstatin A, 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 (5 ml of homogenization buffer/g of liver) in a
Potter-Elvehjem homogenizer with five passes of a motorized Teflon
pestle to achieve a 20% homogenate and then filtered through a 50-µm
nylon mesh to remove fibrous and undisrupted tissue. A 10-ml aliquot of
homogenate was layered onto a discontinuous gradient of 11 ml In studies where PEG precipitation, HPLC, and gel permeation
chromatography of 125I-[A14]insulin were under
investigation, endosomes were prepared in homogenization and gradient
buffers containing 10 mM HEPES buffer, pH 7.4, 2.5 mM N-ethylmaleimide (NEM), 1 mM
1,10-phenanthroline, and 1 mM chloroquine. The
homogenization buffer to prepare samples for HPLC analysis additionally
contained 60 units/ml bacitracin.
Plasma membrane and endosomal fractions (0.5 ml) were
solubilized in 0.5 ml of freshly prepared 2 × solubilization
buffer (50 mM HEPES buffer, pH 7.4, containing 1% Triton
X-100, 150 mM NaCl, 10 mM EDTA, 10 mM sodium pyrophosphate, 1 mM sodium
orthovanadate, 30 mM NaF, 0.5 mM
phenylmethanesulfonic fluoride, 2.5 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml antipain: final
concentration). After incubation for 10 min at 4 °C, the samples
were clarified by centrifugation in an Eppendorf centrifuge at
13,000 × gav (12,000 rpm, 5 min,
4 °C).
Tyrosine kinase activity was assessed
using a modified microtiter plate immunocapture method as described
previously (38, 39). Fifty microliters of CT-3 in 20 mM
NaHCO3, pH 9.6, at a concentration of 0.5 µg/ml was
incubated for 16 h at 4 °C in 96-well F16 Maxisorb loose
Nunc-immuno microwell module plates. Any unattached antibody was
removed with three applications (100 µl) of wash buffer (50 mM HEPES buffer, pH 7.4, 150 mM NaCl, 0.1%
BSA, 0.1% Triton X-100, and 0.1% Tween 20). Duplicate solubilized
plasma membrane or endosome samples (200 µl) were added to wells and incubated for 16 h at 4 °C. The sample was then aspirated and plates washed three times with ice-cold wash buffer (100 µl) and 20 µl of kinase reaction mixture applied (130 mM HEPES, pH
7.4, 100 µg/ml BSA, 1 mM EGTA, 12 mM
MgCl2, 0.2 µM FYF, and 74 KBq [ Solubilized plasma membrane and endosomes
were incubated with microtiter plates as described above in the kinase
assay up to the point of kinase reaction mixture addition, except that in the case of phosphotyrosine content estimation, the wells were plated with phosphotyrosine antibody 4G10 at a concentration of 1 µg/ml instead of CT-3 and the sample volume was 100 µl. Following aspiration of the sample and three washes with wash buffer, the bound
antibody was incubated for 16 h at 4 °C with 50 µl of binding buffer (100 mM HEPES buffer, pH 7.4, 120 mM
NaCl, 1 mM EDTA, 15 mM sodium acetate, 1.2 mM MgSO4, 10 mM glucose, and 1%
BSA) containing 333 Bq of 125I-[A14]insulin. The wells
were then aspirated and washed three times with 100 µl of PBS, pH
7.4, and the bound insulin released by the addition of 100 µl of
0.03% SDS in water for 20 min at room temperature. The 100 µl was
removed from the wells and counted for radioactivity in an LKB 1282 Following hepatic portal vein injection
of 125I-[A14]insulin (1 MBq in 0.5 ml of PBS, 5%
RIA-grade BSA), endosomes were prepared by discontinuous sucrose
gradient centrifugation as described above. The isolated endosomal
population was subsequently washed free of cytosol by a 10-fold
dilution in 10 mM HEPES buffer, pH 7.4 (containing 0.1 M KCl, 2.5 mM NEM), and sedimented by
centrifugation in a Sorvall T-865.1 rotor at 100,000 × gav (37,000 rpm, 30 min, 4 °C). The endosomes
were solubilized with a final concentration of 0.1% Triton X-100 at
4 °C for 10 min and then clarified by centrifugation in a Beckman
TLA 100.2 rotor at 120,000 × gav (60,000 rpm,
10 min, 4 °C). HPLC separation of extracted endosomal contents was
performed at a flow rate of 1 ml/min, in 0.1 M ammonium
acetate buffer, pH 5.5, with an acetonitrile gradient. The precise
changes in acetonitrile concentration required to effect the separation of degradation intermediates were as follows: 1) 0-5 min at 0%, 2)
5-20-min gradient rising to 8.45%, 3) instantaneous rise to 28.5%,
4) 20-40-min gradient rising to 32.5%. Detection of 125I
was achieved by an on-line counter attached to a reverse phase HPLC
Hypersil-BDS, 5 µM, C-18 column, 250 × 4.6 mm,
fitted with an additional 10-mm guard cartridge of the same material
and purchased from Shandon HPLC (Runcorn, Cheshire, UK).
Endosomes
containing 125I-[A14]insulin were prepared as described
above from animals that had received an injection of
125I-[A14]insulin (1 MBq in 0.5 ml of PBS, 5% RIA-grade
BSA) via the hepatic portal vein. The endosomes were solubilized with a final concentration of 0.1% Triton X-100 at 4 °C for 10 min and then clarified by centrifugation in a Beckman TLA 100.2 rotor at
120,000 × gav (60,000 rpm, 10 min,
4 °C). A 0.25-ml aliquot of the supernatant was mixed with 0.5 ml of
bovine A 0.5-ml aliquot of
solubilized endosomes containing 125I-[A14]insulin
prepared as above was applied to a Sephadex G-50 column (40 × 1 cm) equilibrated with 50 mM HEPES buffer, pH 8.0, containing 17 mM NaCl, 2.5 mM NEM, 1.0 mM 1,10-phenanthroline, 1 mM chloroquine, 0.5%
BSA. Elution was achieved with the same buffer, and 1-ml fractions were
counted for radioactivity using LKB 1282 Aliquots (250 µl) derived from endosomes containing
125I-[A14]insulin were added to 500 µl of ice-cold 20%
trichloroacetic acid and incubated on ice for 10 min prior to
centrifugation in an Eppendorf centrifuge at 13,000 × gav (12,000 rpm, 5 min, 4 °C). The pellet and
supernatant were then counted for radioactivity using an LKB 1282 Animals were treated with either PBS or chloroquine as
described above prior to a 0.15 µg/100 g body weight insulin
injection via the jugular vein, which also contained
[14C]glucose (46.3 KBq/100 g body weight). One hour
later, the animals were sacrificed, and their diaphragms excised,
rinsed in ice-cold PBS and blotted dry prior to weighing. The extent of
[14C]glucose incorporation into glycogen was determined
by the method of Rafaelsen et al. (40).
Protein content was determined by a Coomassie
Blue dye binding kit (Pierce) using BSA as standard.
Analyses were performed using the
statistical program SPSS version 7. For all data sets, it was first
determined that the data was normally distributed and that the
variances were equal. These criteria were satisfied in all cases
allowing the use of Student's t test to analyze for
significant differences. Differences in integrated response curves were
tested by two-way analysis of variance (ANOVA) (41).
It has been shown previously
that hepatic insulin degradation in vivo occurs within
endosomes (12), with dissociation of insulin from the receptor being a
prerequisite for this process to occur (14, 17). To determine the
percentage of insulin in endosomes that is receptor-bound, rats were
administered 125I-[A14]insulin via the hepatic portal
vein and hepatic endosomes freshly isolated. Fig.
1 shows the elution profile from a G-50 gel permeation column of material generated from solubilized hepatic endosomes. The first peak represents receptor-bound insulin, which is
size-excluded from the column and elutes in the void volume. This
comprised 18.5% of the total. The amount of receptor-bound insulin was
confirmed by an independent method of assessment, namely PEG
precipitation of the insulin-receptor complex (19.2%; Table
I). The remaining 80% of the insulin
eluted in the second peak from the G-50 column and comprised
dissociated insulin, of which 86.3% remains intact as assessed by
trichloroacetic acid precipitation (Table I). Thus, after insulin
injection, 2 min of in vivo processing time, and endosome
preparation time, 80% of endocytosed insulin has already dissociated
from its receptor and is accessible for intra-endosomal
degradation.
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Table I.
Effect of chloroquine on insulin-receptor interactions and inhibition
of endosomal insulin degradation in vivo
Volume 272, Number 43,
Issue of October 24, 1997
pp. 26833-26840
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Chloroquine Extends the Lifetime of the Activated Insulin
Receptor Complex in Endosomes*
,
-subunit and the activation of the receptor tyrosine
kinase toward exogenous substrates (1, 2). Following
autophosphorylation, the activated ligand-receptor complex is
internalized into endosomes in liver (3-6) and low density membranes
in adipocytes (7, 8) and muscle (9). Endocytosis of activated receptors
has the twin effects of concentrating receptors within endosomes and
allowing the insulin receptor tyrosine kinase to phosphorylate
substrates that are spatio-temporally distinct from the plasma membrane
(Ref. 10; reviewed in Ref. 11). Subsequent termination of signal
transduction is achieved by endosomal insulin degradation (12-16)
following dissociation of insulin from its receptor (14, 17) as the
intralumenal environment of the endosome acidifies (18). This loss of
the ligand-receptor complex attenuates any further ligand-driven
receptor re-phosphorylation, and the receptor is dephosphorylated by
extralumenal endosomally associated phosphotyrosine phosphatase(s) (5,
6, 19).
Animals
-32P]ATP (9 TBq/µmol) were
from Amersham International (Bucks., UK). Metafane anesthetic was from
C-VET Ltd. (Bury St. Edmonds, Suffolk, UK), and Sagital (sodium
pentabarbitone) was from May and Baker Ltd. (Dagenham, UK).
Polyethylene glycol 6000 (PEG-6000; molecular weight 6000-7500) was
from BDH (Poole, Dorset, UK). The synthetic peptide FYF (RDIFEADYFRK)
was synthesized commercially and corresponds to residues based on the
kinase regulatory domain of the insulin receptor (32) except that two
tyrosines have been replaced with phenylalanine to afford a higher
degree of linearity to the kinase assay.
-subunit was used for immunocapture assays. CT-3 was prepared using
as antigen a GST-fusion protein containing the terminal 100 amino acids
of the human insulin receptor (36) and purified as described previously
(37).
= 1.09 g·cm
3 (0.67 M) and 12 ml
= 1.14 g·cm
3 (1.05 M) sucrose in 33-ml centrifuge
tubes and centrifuged in a Sorvall AH-629 swing-out rotor (Du Pont) at
110,000 × gav (29,000 rpm, 75 min,
4 °C) in a Sorvall OTD75B centrifuge (Du Pont). Endosomes were
collected at the interface of the 1.09 and 1.14 g·cm
3
sucrose solutions.
-32P]ATP). The incubation was terminated after 20 min
at room temperature by the addition of 5 µl of 2 M HCl.
32P-Labeled peptide substrate was captured onto
phosphocellulose columns (SpinzymeTM, Pierce, Chester, UK) by
centrifugation in an Eppendorf centrifuge at 13,000 × gav (12,000 rpm, 0.5 min, 22 °C). The filter
was washed twice with 75 mM phosphoric acid (500 µl) and
centrifuged as before. Finally, the filter cartridge was removed and
counted in 10 ml of scintillation mixture in a Canberra Packard 1500 Tri-Carb liquid scintillation analyzer.
-counter.
-globulin (1.25 mg/ml PBS, pH 7.4) and 0.5 ml of PEG-6000
(25% in water). The mixture was left for 20 min at 4 °C and then
centrifuged in a Jouan KR 422 centrifuge at 3000 × gav (3200 rpm, 25 min, 4 °C). The pellet was then
counted for radioactivity using an LKB 1282
-counter.
-counter.
-counter.
Determination of the Amount of Receptor-bound Insulin in Freshly
Isolated Hepatic Endosomes and the Effect of Insulin Dissociation on
Subsequent Endosomal Degradation
Fig. 1.
Determination of the amount of receptor-bound
insulin present in freshly isolated endosomes. Anesthetized rats
were injected with approximately 1 MBq of
125I-[A14]insulin via the hepatic portal vein over
30 s. The liver was removed after an additional 2 min and
endosomes prepared as described under "Experimental Procedures."
Inhibitors of insulin degradation were omitted from the sucrose
solutions. Endosomes were disrupted and extracted into 0.1% Triton
X-100 and applied to a G-50 Sepharose column in the presence of 2.5 mM NEM, 1 mM 1,10-phenanthroline, and 1 mM chloroquine. Fractions (1 ml) were collected and counted
for radioactivity. The figure is representative of two experiments from
two separate animals.
) inhibitors). Endosomes were then disrupted and extracted into 0.1% Triton X-100 and applied to either a G-50 Sepharose column in the presence of 2.5 mM NEM, 1 mM 1,10-phenanthroline, and 1 mM chloroquine or
subjected to trichloroacetic acid and PEG precipitation. Values
represent the mean and S.E. from two to three separate animals. *,
effect of chloroquine on the percent of insulin bound in the presence
of buffer protease inhibitors (p < 0.02).
Treatment
Trichloroacetic acid
precipitability
PEG precipitability
Sepharose G-50
chromatography
%
%
%
(
)
Inhibitors86.3
± 5.5
19.2 ± 2.7
18.5
(
)
Chloroquine
(+) Inhibitors
97.5
± 0.4
27.7 ± 1.6
29.7
(
) Chloroquine
(+) Inhibitors
98.9 ± 0.2
52.6
± 4.1*
57.3
(+) Chloroquine
A previous study (28) had shown chloroquine capable of enhancing the
association of insulin to its receptor. The effect of chloroquine
in vitro was examined on insulin-receptor dissociation and
degradation. Hepatic endosomes, containing
125I-[A14]insulin internalized over a 2-min time period,
were incubated for 1 h at 25 °C with concentrations of
chloroquine ranging from 0.5 to 5 mM. In the absence of
chloroquine, a 1-h incubation resulted in 18.5% of the insulin
remaining receptor-associated (Fig.
2B) and 56.5% degraded (Fig.
2A). Inclusion of chloroquine in the incubation buffer
augmented the amount of receptor-associated insulin to a maximum of
28% (p < 0.05) in the presence of 5 mM chloroquine with only 16.3% now being in a degraded state
(p < 0.01). Thus, this in vitro experiment
confirmed the need for insulin to dissociate from its receptor for
subsequent degradation, and the inhibitory role played by chloroquine
in this process.
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The role(s) of chloroquine were next examined under more physiological
conditions in vivo. For this study, chloroquine was administered intraperitoneally prior to 125I-[A14]insulin
administration. Endosomes were prepared and solubilized and their
contents separated on a G-50 gel permeation column as in Fig. 1, except
that protease inhibitors were present in buffers at all stages of
endosome preparation and separation. Fig.
3A shows animals that received
only injection of vehicle (PBS) 2 and 1 h prior to
125I-[A14]insulin injection. The first peak, as in Fig.
1, comprises receptor-bound insulin and constitutes 29.7% of the
label, while in vivo chloroquine treatment (Fig.
3B) increased this figure to 57.3%. PEG precipitation
confirmed these findings with values of 27.7% and 52.6%, respectively
(p < 0.02) (Table I). Additionally, inclusion of
protease inhibitors in all preparative steps decreased degradation of
label from 13.7% (in their absence) to 2.5% (with their inclusion)
(Table I). Since trichloroacetic acid precipitability is not a
definitive index of insulin integrity, HPLC separation of the
endocytosed insulin was also performed on the samples. Fig.
4A depicts the elution profile
in the absence of chloroquine treatment in vivo. The peak
corresponding to 125I-[A14]insulin constituted only 46%
of the total, while chloroquine treatment increased this value to 89%
(Fig. 4B). Thus, in vivo chloroquine treatment
markedly increased the percentage of insulin remaining intact following
endocytosis into hepatic endosomes, which in turn is due in part to its
ability to decrease insulin dissociation from its receptor.
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The Effect of Chloroquine on Endosomal Insulin Receptor Signal Transduction
Having established the ability of chloroquine to potentiate insulin binding to its receptor and inhibit degradation within endosomes, its effect on insulin signal transduction was next examined. Animals were injected intraperitoneally with either PBS or chloroquine 2 and 1 h prior to insulin administration and sacrificed between 0 and 45 min later. Following preparation of hepatic endosomes the amount of insulin receptor, its phosphotyrosine content and tyrosine kinase activity were established using microtiter plate capture assays.
The flux of insulin receptors through the endosomal compartment
following insulin administration in vivo, as determined by immunocapture, is shown in Fig. 5. The
data are normalized so that the basal value of the control group is set
to 100%. In the absence of chloroquine, the control group showed a
rapid doubling of endosomal insulin receptor content by 5 min and
return to basal values by 18 min. Following chloroquine treatment, the
integrated response for endosomal receptor content was increased
throughout the time course by 31% (p < 0.05). Even at
45 min after insulin administration, the endosomal insulin receptor
content had not returned to control basal levels, indicative of
chloroquine-sensitive inhibition of receptor recycling. Receptor
phosphotyrosine content (Fig. 6)
demonstrated a similar time course, rapidly increasing by 5 min after
insulin injection and remaining elevated at 10 min until return to
basal level at 18 min. Chloroquine treatment augmented the
phosphotyrosine content, leading to a 64% increase in the integrated
response-time curve (p < 0.01). Finally, the most
striking effect of chloroquine was observed with the insulin receptor
tyrosine kinase activity toward an exogenous substrate (Fig.
7). Tyrosine kinase activity reached a
rapid peak of 300% over basal values at 5 min after insulin injection,
remained elevated at 10 min, and returned to base line by 18 min.
Chloroquine treatment substantially augmented the tyrosine kinase
activity throughout the time course, giving a 96% increase in the
integrated response (p < 0.001). Thus, chloroquine
potentiated the normal insulin-induced receptor signal both in its
magnitude and duration.
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In contrast, chloroquine was without observable effect on either the phosphotyrosine content or the tyrosine kinase activity of insulin receptors situated in the plasma membrane. This was assessed at both peak activation for the plasma membrane-located receptor (0.5 min after insulin injection (6); Table II) and during the 45-min time course (data not shown). Therefore, these data suggest that the effect of chloroquine on the insulin receptor is restricted to the endosomal compartment.
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Chloroquine did not appear to inhibit phosphotyrosine phosphatases associated with the endosomal insulin receptor since receptor phosphotyrosine content, expressed per unit of insulin receptor, was not significantly increased with chloroquine treatment throughout the time course (p = 0.299) (Table III). This contrasts with previous studies, where specific phosphotyrosine phosphatase inhibitors increased phosphotyrosine content greater than 5-fold (6). The exogenous tyrosine kinase activity of the insulin receptor demonstrated a significant increase in the presence of chloroquine (p < 0.05) (Table III). This increase in tyrosine kinase activity is most likely due to a continued association of insulin with its receptor in the presence of chloroquine.
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In light of the above evidence for a
chloroquine-dependent enhancement of insulin signal
transduction, the incorporation of glucose into muscle glycogen was
assessed in the absence and presence of chloroquine. Rats were treated
with PBS or chloroquine as before prior to intrajugular administration
of insulin and tracer [14C]glucose. After an additional
1 h, the animals were sacrificed and the incorporation of glucose
into glycogen in diaphragm was assessed. Fig.
8 shows that insulin stimulated
incorporation of glucose into glycogen in a manner similar to that
detailed in previous reports (42). In vivo chloroquine
treatment significantly augmented the insulin effect by 145%
(p < 0.001) and demonstrated at the level of a
metabolic response the potentiation of insulin signaling by
chloroquine.
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Previous studies have shown that subsequent to insulin administration in vivo, hepatic insulin receptors bind insulin at the plasma membrane, followed by very rapid internalization of receptor (3-5) and bound insulin (43, 44) into endosomes where the number of receptors increases approximately 5-fold (5, 6). It has been demonstrated that the endosomal insulin receptor tyrosine kinase remains tyrosine-phosphorylated and is even more active than its receptor located within the plasma membrane (4-6). The selective activation of the endosomal insulin receptor kinase, by the phosphotyrosine phosphatase inhibitor bisperoxovanadium phenanthroline, in the absence of insulin, has been shown to lead to phosphorylation of IRS-1 and to elicit a hypoglycemic response in vivo (10, 11). Comparable studies of the hepatic epidermal growth factor and its receptor have revealed a similar phenomenon (45-47) where endosomes containing activated ligand-bound receptors constitute the major locus for Shc tyrosine phosphorylation (48, 49). Together, these findings have prompted the suggestion that endosomes play a pivotal role in signal transduction, giving the receptor tyrosine kinase access to substrates distal to and topographically distinct from the plasma membrane (11). In this study, we have demonstrated that in vivo administration of chloroquine, a compound that inhibits both dissociation of insulin from its receptor (28) and subsequent endosomal degradation of insulin (12), leads to a prolongation of the activated insulin-receptor complex and augments insulin-stimulated responses. A key observation is that the in vivo locus for both the inhibition of insulin dissociation and degradation by chloroquine is restricted almost entirely to the endosomal compartment (Figs. 1, 2, 3, 4). These findings are in agreement with previous in vitro studies, where it was observed that chloroquine augmented the binding of insulin to its receptor in purified rat liver plasma membrane preparations (28) and inhibited endosomal insulin degradation in isolated vesicles (12).
In the present study, chloroquine was without apparent effect on insulin receptors present in the plasma membrane (Table II). However, differences in conditions between the studies may explain the apparent discrepancy. The concentration of chloroquine required to demonstrate the phenomenon in vitro were in the order of 1 mM, whereas in this study the blood concentration of chloroquine was in the low micromolar range (data not shown) and thus could not elicit an effect on the receptors in the plasma membrane. However, since chloroquine is an acidotrophic agent and accumulates in acidic environments such as insulin-containing endosomes (18) and lysosomes (50), high millimolar concentrations are rapidly achieved within these organelles (23) allowing the effect to occur within endosomes. In addition, since insulin's interaction with the plasma membrane receptor is very transient (<30 s) before entry into endosomes, its window of opportunity for an effect is very limited. Thus, the effect of chloroquine on insulin dissociation and degradation in vivo is restricted to endosomes.
The findings that in vivo chloroquine treatment inhibited insulin dissociation from its receptor and reduced insulin degradation led us to investigate whether in vivo chloroquine treatment could augment the insulin signaling cascade from this compartment as hypothesized previously (28). The flux of insulin receptors through the endosomal compartment of the liver following insulin administration was significantly increased in amount and duration in the presence of chloroquine (Fig. 5). This increased accumulation suggested either that chloroquine was inhibiting recycling of receptors back to the plasma membrane following their insulin-induced endocytosis or that insulin receptors were endocytosing more rapidly in the presence of chloroquine. The retention of receptors within the endosome might result from sustained receptor tyrosine phosphorylation. Dephosphorylation has been suggested as a possible signal required before return of receptors to the plasma membrane can be effected (51, 52), and endosomal insulin receptors have been shown to be specifically dephosphorylated by phosphotyrosine phosphatase(s) prior to their return to the plasma membrane (6, 19). Such a mechanism would require sustained endosomal insulin receptor tyrosine phosphorylation following chloroquine treatment. This was demonstrated by the data in Fig. 6, where insulin-induced receptor tyrosine phosphorylation was shown to be augmented by chloroquine throughout the time course. In a similar manner, the endosomal insulin receptor exogenous tyrosine kinase activity was also increased (Fig. 7). Therefore, the chloroquine-augmented insulin-receptor complex formation within endosomes not only prolonged the residency of the insulin receptor within the endosomal compartment but enhanced its activation in both duration and amount. Enhanced interaction of insulin with its receptor leading to sustained receptor tyrosine phosphorylation and signaling has also been observed using analogues of insulin whose binding characteristics are greatly augmented (53).
Consequential to the sustained endosomal activation of the insulin receptor, assessment of chloroquine's effect on an insulin-dependent event, in vivo incorporation of glucose into glycogen in rat diaphragm (40, 42) resulted in an augmentation of glycogen synthesis (Fig. 8). This response was determined in diaphragm, since it is not easily shown in rat liver due to the glucose paradox (54). Additionally, in another study, chloroquine resulted in a pronounced improvement in glycemic control following an oral glucose challenge in rats.3 These observations correlated well with effects in human non-insulin-dependent diabetes mellitus patients treated with chloroquine. Here, improvement in glucose tolerance (29), increased peripheral glucose disposal, and decreased metabolic clearance rates of insulin (30) were observed. In insulin-dependent diabetes mellitus patients, chloroquine has been shown to reduce insulin resistance (31). These augmentations of insulin-induced metabolic effects all appear consequential to a prolongation of the effective half-life of insulin in endosomes as described in this study. Indeed, in an investigation of rat adipocytes where the endosomal insulin receptor tyrosine kinase is known to be active (7, 8), IRS-1, the distribution of which was 20% endosomal and 80% cytosolic, demonstrated that its tyrosine phosphorylation paralleled that of the receptor tyrosine kinase in the endosome (8). Consequently, a strong case for a role of the activated endosomal insulin receptor has been made in the recruitment of the insulin-sensitive GLUT 4-containing vesicles from their intracellular depot to the plasma membrane following insulin stimulation in adipocytes (8, 55).
In summary, these observations are consistent with a
chloroquine-dependent inhibition of endosomal
insulin-receptor dissociation and subsequent degradation, sustaining
the half-life of the active endosomal receptor and potentiating insulin
signaling from this compartment as depicted in Fig.
9. Additionally, it demonstrates the
usefulness of chloroquine as research tool to help elucidate the role
endosomes in signal transduction.
[View Larger Version of this Image (41K GIF file)]
To whom correspondence should be addressed: Dept. of Clinical
Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills
Road, Cambridge CB2 2QR, United Kingdom. Tel.: 44-1223-336793; Fax:
44-1223-330598; E-mail: apb28{at}cam.ac.uk.
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