 |
INTRODUCTION |
The pancreatic
-cell plays a key role in glucose
homeostasis by secretion of the hormone insulin. The first step in
insulin secretion is the metabolism of glucose in
-cells (1-3).
Glucose enters the
-cell through GLUT2 transporters on the plasma
membrane. Glucose metabolism begins with glucokinase, the
-cell
glucose sensor (4), and results in an increase in the intracellular ATP/ADP ratio. This causes closure of ATP-dependent
K+ channels and
-cell plasma membrane depolarization.
The depolarization event leads to Ca2+ influx through
voltage-gated L-type Ca2+ channels (5, 6). Increased
cytosolic Ca2+ stimulates insulin exocytosis by the
-cell through Ca2+-dependent protein kinase pathways.
Insulin acts on a variety of tissues by binding to the insulin receptor
(IR).1 Insulin binds to the
dimerized insulin receptor and causes activation of the catalytic
tyrosine kinase in the IR
-subunit. The IR tyrosine kinase then
autophosphorylates intracellular tyrosine residues on the insulin
receptor itself, which act as docking sites for insulin receptor
substrates 1 and 2 (IRS-1 and IRS-2) and Shc. These proteins are then
phosphorylated by the insulin receptor and interact with several
downstream effectors including phosphoinositide 3-kinase and Grb2.
These effectors mediate glucose transport, cell growth, and various
other important cellular functions.
A growing body of evidence has confirmed that the insulin
receptor-signaling pathway is active in the pancreatic
-cell and is
involved in regulating key cellular processes (7-16). Insulin stimulation of the
-cell IR results in tyrosine phosphorylation of
the catalytic IR
-subunit and IRS proteins (8). Mice with a
pancreatic
-cell-specific knockout of IR exhibit hyperinsulinemia and impaired glucose tolerance that develops after 6 months (11). In
addition, these mice lose their acute first-phase glucose-stimulated insulin secretion response. The loss of IRS-1 leads to mild insulin resistance, hyperinsulinemia, and
-cell hyperplasia but no overt diabetic phenotype (17-20). Islets and
-cells derived from these IRS-1 knockout mice show decreased insulin content of 51 and 55%, respectively, and a lower glucose-stimulated insulin secretion response
(21). In contrast, the loss of IRS-2 in mice causes early insulin
resistance and eventually a decrease in
-cell mass,
-cell
failure, and overt type II diabetes (17, 22). Analysis of islets and
-cells from IRS-2 knockout mice revealed no secretory defects, but
altered
-cell growth (23). This difference in phenotype of IRS
knockouts indicates that these substrates mediate different functions
in the
-cell. The mechanisms by which IRS-1 and IRS-2 accomplish
these roles remain unclear.
Two studies that examined the role of IRS-1 in insulin secretion from
isolated pancreatic
-cells and
-cell lines found that IRS-1 also
has an effect on calcium homeostasis. In one study, the overexpression
of IRS-1 in the
TC6-F7 cell line (
-IRS1) lead to an increase in
the glucose-stimulated fractional insulin secretion (ratio of secreted
insulin/total insulin content) and, interestingly, an increase in
cytosolic Ca2+ (7). This increase in cytosolic
Ca2+ was also seen in a cell line overexpressing the IR,
but not in a cell line overexpressing a kinase-deficient mutant of IR.
Further investigation revealed that the increase in cytosolic
Ca2+ was the result of inhibition of the sarco-endoplasmic
reticulum Ca2+-ATPase (SERCA), a protein responsible for
Ca2+ uptake into the ER lumen. When the control
-cell
line was treated with thapsigargin, a SERCA inhibitor, the cytosolic
Ca2+ levels increased to the same level as the
-IRS1
cell line. Furthermore, ER calcium uptake in a digitonin-permeabilized
cell system was reduced in the
-IRS1 cells compared with controls.
In another study, insulin exocytosis and intracellular Ca2+
levels of single
-cells from IRS-1-deficient mouse islets and IRS-1
null
-cell lines were measured (24). In the absence of IRS-1,
insulin exocytosis and cytosolic calcium were decreased. Taken
together, these data indicated that some mechanism mediated by the
activation of IRS-1 through the insulin receptor affects the influx of
Ca2+ into the ER, resulting in changes in Ca2+
homeostasis and insulin secretion. One potential explanation for this
effect is that IRS-1 interacts directly with SERCA and inhibits its
function. For this to occur, activated IRS-1 must first be in the same
vicinity as SERCA.
Previous studies examining the subcellular distribution of IRS proteins
in 3T3-L1 adipocyte cell lines have shown that both IRS-1 and IRS-2
distribute to the cytosol and intracellular membranes (IM) of cells,
but the majority is located within the IM (25-33). Studies using
ultracentrifugation, sucrose density gradients, and detergent
precipitation, demonstrate that IRS proteins localize to a high speed
pellet (HSP) fraction that contains the endoplasmic reticulum and the
trans-Golgi network (31-37). Upon stimulation with insulin, IRS-1 is
preferentially tyrosine-phosphorylated in the intracellular membrane
compartment, where it remains for several minutes before translocating
to the cytosol (32). The distribution of IRS-1 to the same
intracellular membrane as SERCA, namely the ER, lends credence to the
idea that these two proteins can interact. In addition, there is
evidence for a direct interaction of IRS and SERCA proteins. In rat
muscle extracts, IRS-1 and IRS-2 were shown to co-immunoprecipitate
with SERCA1 and SERCA2a, the skeletal and cardiac muscle isoforms of
SERCA (38). Insulin stimulation of the rats resulted in a 2-6-fold
increase in the association of IRS proteins with SERCA. IRS-1 and IRS-2
also bound to the ubiquitously expressed SERCA2b isoform, which is also
co-expressed with SERCA3 in pancreas (39, 40).
In this current study, we have employed several different techniques to
show that IRS-1 and IRS-2 proteins localize to intracellular membranes
in pancreatic
-cell lines. More specifically, IRS-1 co-localizes
with SERCA3b, one of two SERCA isoforms present in
-cells, to
endoplasmic reticulum-derived microsomes prepared from
-cells.
SERCA3b has also been shown to co-immunoprecipitate with IRS-1 in a
model cell line and
-cell lines. This interaction is enhanced with
acute insulin stimulation of the cells. Examination of in
vitro ER Ca2+ uptake in both wild-type and
IRS-1-overexpressing
-cells demonstrates that the
-cell SERCA
isoforms function normally and respond to inhibition with thapsigargin
in the same fashion. Finally, treatment of wild-type
-cells with
thapsigargin resulted in an increase in glucose-stimulated fractional
insulin secretion comparable with the increase observed for untreated
IRS-1-overexpressing
-cells. These results provide a functional link
between the direct IRS-1/SERCA interaction and regulation of calcium
homeostasis and insulin secretion.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines and Culture Media--
The
-IRS1 cell line and
clonal mouse
-cell line
TC6-F7 were cultured as previously
described (7). In brief, cells were maintained in high glucose DMEM (25 mM glucose; Invitrogen) supplemented with 10% fetal
bovine serum (HyClone, Logan, UT), 100 units/ml penicillin, 50 µg/ml streptomycin (complete DMEM) and incubated at 37 °C
in a 10% CO2, 90% air humidified incubator. Both cell lines were passaged every 7-10 days and seeded in new T-175 flasks (Falcon, catalog no. 353112) at a cell density of 1:10 (1.0-2.0 × 106 cells/T-175 flask) as follows. The cell lines were
washed with 10 ml of Versene and then incubated with 2 ml of 0.05%
trypsin-EDTA (Invitrogen, catalog no. 25300-054) for 5 min at 37 °C
in a 10% CO2, 90% air humidified incubator. The cells
were then rinsed with complete DMEM, pelleted, and re-suspended in
complete DMEM. Cells were counted and seeded into T-175 flasks as
described above. The Chinese hamster ovary-T cell line (CHO-T), which
stably expresses the human insulin receptor, was a kind gift from Dr.
R. Roth (Stanford University School of Medicine, Stanford, CA). This
cell line was cultured in F-12 (Ham's) medium (Invitrogen)
supplemented with 5% fetal bovine serum, 100 units/ml penicillin, 50 µg/ml streptomycin (complete F-12) and incubated at 37 °C in a 5%
CO2, 95% air humidified incubator.
Expression Plasmids--
The pMT2-SERCA3b expression plasmid was
a kind gift from Dr. F. Wuytack (Laboratorium voor Fysiologie,
Katholieke Universiteit Leuven, Leuven, Belgium). The pCMV-IRS1 was
constructed as described previously (7).
Production of Anti-SERCA3b Antibody--
A rabbit polyclonal
antibody directed against the C terminus of SERCA3b was prepared as
follows. A C-terminal peptide of SERCA3b (sequence = C-TGKKGPEVNPGSRGES) that is unique to the SERCA3b splice
variant was synthesized by the Howard Hughes Medical Institute Biopolymer Facility at Yale University (W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven, CT)
containing an additional N-terminal cysteine (in bold). The peptide was
conjugated to the Imject® maleimide-activated
SuperCarrier® modulator (Pierce catalog no. 77656)
according to the protocol from the manufacturer. The peptide conjugate
was then injected into 2 rabbits (Covance Research Products, Inc.) at
an initial dose of 0.5 mg followed by a booster dose of 0.25 mg every
21 days for the duration of the study. Bleeds were collected 10 days after each booster dose, and the sera were shipped frozen to our laboratory. The sera were screened for antibody production by Western
blot analysis using CHO-T cell lysate from cells transfected with or
without the pMT2-SERCA3b plasmid (described below) as positive and
negative controls. Sera from bleeds containing SERCA3b antibody were
affinity-purified using the Sulfo-Link® kit (Pierce
catalog no. 44895) as follows. The SERCA3b peptide antigen was
dissolved in Sulfo-Link® Coupling buffer (50 mM Tris, 5 mM EDTA-Na, pH 8.5) and coupled to a
column containing Sulfo-Link® Coupling gel via stable
disulfide bonding. Excess free sulfhydryl groups were blocked with the
addition of 0.05 M cysteine in Coupling buffer to the
column. After washing away excess peptide and cysteine, SERCA3b
antibody-containing serum was applied to the column and incubated at
room temperature for 1 h. The column was washed, and then the
SERCA3b antibody was eluted with 100 mM glycine, pH 2.75. Fractions (1 ml) were collected and neutralized by adding 50 µl of 1 M Tris, pH 9.5. Fractions containing the SERCA3b antibody (determined by A280 measured
spectrophotometrically and Bio-Rad protein assay) were pooled and
dialyzed in PBS with 0.05% sodium azide overnight. The concentration
of the SERCA3b antibody solution was determined by Bio-Rad protein
assay and A280 measurements. The solution was
aliquoted and frozen at
20 °C.
Subcellular Fractionation of
-Cell Lines--
Subcellular
fractions were prepared using a modification of the method described by
Colca et al. (41). Both the
TC6-F7 and
-IRS1 were
seeded on 15-cm dishes (Falcon, catalog no. 353025) at a cell density
of 2.0 × 106 cells/dish and cultured for 7 days as
described above. Cells were homogenized on ice in 50 mM
MES, pH 7.2, 250 mM sucrose, 1 mM EDTA buffer
(Fractionation buffer). The homogenate was centrifuged at 600 × g for 5 min to yield pellet PM, containing plasma membrane, nuclear material, and debris. The supernatant was then centrifuged at
20,000 × g for 20 min to yield a secretory vesicle and
mitochondria-enriched pellet (SV). The resulting supernatant was
centrifuged at 150,000 × g for 90 min to yield a HSP,
enriched with IM and cytoskeleton. The HSP fraction was re-suspended in
fractionation buffer without EDTA. The remaining supernatant, called
CYT, contains cytosol. ER enrichment of the HSP was verified by
comparing the activity of the ER marker enzyme, NAPDH cytochrome
c reductase, in the HSP to that in the homogenate. Results
indicate a 4-fold enrichment in ER marker enzyme activity (Table
I).
Immunoblotting of
-Cell Fractions for IRS
Proteins--
Subcellular fractions were analyzed by Western blot
using rabbit polyclonal anti-IRS-1 (Upstate Biotechnology Inc. (UBI)
catalog no. 06-248), anti-IRS-2 (UBI catalog no. 06506), and anti-GLUT2 (H-67) (Santa Cruz catalog no. SC-9117) antibodies. Briefly, 40 µg of
each fraction and the homogenate were run on 7.5% SDS-PAGE gels and
transferred to nitrocellulose. The blots were incubated in a
Tris-buffered saline (TBS-T) blocking buffer (1% bovine serum albumin
(BSA) in 10 mM Tris-Cl, pH 7.5, 100 mM NaCl,
0.1% Tween 20) for 1 h. The blots were then incubated in 1:500
solutions of anti-IRS-1 or anti-IRS-2 antibody in blocking buffer
overnight and washed with TBS-T five times for 5 min each the following day. Next, the blots were incubated for 1 h with
125I-labeled Protein A (Amersham Biosciences, catalog no.
IM144) diluted in blocking buffer. Blots were then washed with TBS-T five times for 5 min each and air-dried. The radiolabeled blots were
exposed on a phosphor screen (Amersham Biosciences) overnight and analyzed with a PhosphorImager scanner.
IRS Protein Localization by Immunofluorescence in
-Cells--
For immunofluorescence,
-cell lines were seeded on
10-mm poly-D-lysine-coated cover slips in 24-well plates at
a cell density of 5 × 104 cells/well. The cells were
cultured under the conditions described above for 2 days. Cells were
washed twice with 0.5 ml of Dulbecco's phosphate-buffered saline,
fixed with 0.5 ml of ice-cold methanol for 10 min and incubated in
blocking buffer (Dulbecco's phosphate-buffered saline supplemented
with 5% FBS) for 10 min. For ER localization, the cover slips were
incubated for 1 h with 50 µl of the following antibodies at the
indicated dilutions in blocking buffer (5% FBS-PBS): anti-IRS-1
(1:100), anti-IRS-2 (1:100), and anti-BiP/GRP78 (Transduction Laboratories, catalog no. G73320) (1:50). The cover slips were washed
with PBS and then incubated for 1 h with 50 µl of secondary antibody solutions in blocking buffer as follows: goat anti-rabbit labeled with cyanine 2 (cy2) (1:500) and/or donkey anti-mouse labeled
with Texas Red (TR) (1:500) (Jackson Immunoresearch Laboratories, Inc.,
catalog no. 111-225-03 and 715-075-150, respectively). For Golgi
localization, the same procedures were followed substituting anti-GM130
(Transduction Laboratories, catalog no. G65120) for anti-BiP/GRP78 at a
1:50 dilution. Immunofluorescence-labeled cover slips were mounted onto
microscope slides with PermaFluor reagent (Immunon, catalog no. 434980)
and analyzed by confocal microscopy in the University of Pennsylvania
Diabetes Center Biomedical Imaging Core. Images were captured using the
Nikon Eclipse E600 fluorescent microscope and employing a krypton-argon
laser as a light source. The Bio-Rad MRC1024 software was used to
operate the microscope and laser source. Cross-sectional images through the
-cells were collected in a series of slices 0.10-0.30 µm apart (depending on the total diameter of the cells) from the upper to
the lower surface of the cells (~25-40 image slices/cell group).
Each image was collected concurrently at 506 nm (green) and 615 nm
(red). The images were analyzed using Confocal AssistantTM
version 4.02 software (copyright 1994-1996, Todd Clark Brelje).
Immuno-electron Microscopy on IM-enriched
Fractions--
Pre-coated nickel grids were floated on a solution of
the IM-enriched fraction (HSP) from both
TC6-F7 and
-IRS1 cells
for 10 min. The grids were then incubated on 20-µl drops of blocking buffer (1% ovalbumin, 0.2% cold water fish skin gelatin, PBS, pH 7.4)
and washed four times with 50 mM Tris, 20 mM
glycine for 1 min each. The grids were next incubated on 5-µl drops
of the following primary antibody solutions for 60 min: rabbit
polyclonal anti-IRS-1 (1:50 dilution), mouse monoclonal anti-IRS-1
(Santa Cruz, catalog no. sc8038) (1:50), anti-SERCA3b (1:500), and
anti-BiP/GRP78 (1:200). The incubations were performed for each
individual antibody solution alone and in the following combinations:
rabbit polyclonal anti-IRS-1 + anti-BiP/GRP78, anti-SERCA3b + anti-BiP/GRP78, and mouse monoclonal anti-IRS-1 + anti-SERCA3b. Grids
were washed four times with 0.1% BSA in PBS and incubated with the
appropriate combination of species-specific gold-conjugated secondary
antibody solutions in 0.1% BSA, acetylated PBS (Aurion, catalog no.
25558) for 45 min. The gold-conjugated secondary antibodies used were anti-rabbit conjugated to 18-nm gold particles and anti-mouse conjugated to 5-nm gold particles (Rockland, Gilbertsville, PA). The
grids were then washed four times with 0.1% BSA in PBS for 5 min each
and incubated in 1% glutaraldehyde in deionized water for 5 min to
stabilize the signal. The grids were washed with deionized water, fixed
for 5 min in 1% osmium (in deionized water), and rinsed with deionized
water. Finally, all grids were stained with 2% aqueous uranyl acetate
for 5 min and then dried. Grids were examined by electron microscopy at
the University of Pennsylvania Diabetes Center Biomedical Imaging Core
using the JEOL JEM1010 electron microscope (JEOL, Tokyo, Japan). Images
were collected at 80.0 kV and magnification ×120,000.
Co-immunoprecipitation of SERCA3b and IRS-1 Proteins--
CHO-T
cells were seeded in 15-cm dishes and cultured overnight as described
above. When cells were 50-70% confluent, they were transiently
transfected with pMT2-SERCA3b alone or with pMT2-SERCA3b and pCMV-IRS-1
using the FuGENETM 6 transfection reagent (Roche Molecular
Biochemicals, catalog no. 1814443) in a 6:1 FuGENETM 6:
total DNA ratio as follows: 90 µl of FuGENETM 6 and 15 µg of pMT-SERCA3b; 180 µl of FuGENETM 6, 15 µg of
pMT-SERCA3b, and 15 µg of pCMV-IRS-1. Each reagent mixture was enough
to transfect two 15-cm dishes. After 24 h of transfection, cells
were incubated in starvation medium (F-12 medium, 0.1% FBS) for 3 h and then treated for 5 min with or without insulin (100 nM). The cells were lysed with Kasuga's lysis buffer (30) (20 mM Tris-Cl, pH 7.6, 1% Nonidet P-40, 10%
glycerol, 137 mM NaCl, 1 mM MgCl2,
1 mM CaCl2, 20 mM
Na4P2O7, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin and
aprotinin) supplemented with 100 µl/10 ml of phosphatase inhibitor
mixture 2 (Sigma, catalog no. P-5726).
TC6-F7 and
-IRS1 cells
were seeded and cultured to confluence as described above. The cells
were washed twice and then incubated with 20 ml of Krebs-Ringer buffer (KRB) (115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 25 mM Hepes, pH 7.4)
supplemented with 0.1% BSA for 30 min prior to insulin treatment. The
KRB solution was changed, and then the cells were treated with insulin
as described above. The protein concentration of the lysates was
determined by the Bio-Rad BCA protein assay. Sample aliquots of 500 µg were pre-cleared with 20 µl of a 50% Protein A-Sepharose CL-4B
slurry (Amersham Biosciences, catalog no. 71-7090-00). The
samples were incubated overnight with anti-IRS-1, anti-SERCA3b,
pre-immune serum, or normal rabbit serum. Immune complexes were pulled
down with 40 µl of the 50% Protein A-Sepharose CL-4B slurry
overnight, washed twice with Wash Buffer 1 (50 mM Hepes, pH
7.8, 1.0% Triton X-100, 0.1% SDS, 150 mM NaCl), and then
washed twice with Wash Buffer 2 (50 mM Hepes, pH 7.8, 1.0%
Triton X-100, 0.1% SDS). Laemmli buffer (with 3 mg/ml dithiothreitol)
was added to the samples, which were then analyzed by Western blot
using the anti-IRS-1 and anti-SERCA3b antibodies.
ER Ca2+ Uptake Measurements of ER-enriched
Fractions--
Calcium uptake was measured as described previously
(41). 45CaCl2 (ICN Biomedicals, Inc., Catalog
no. 62005) was used as a tracer of calcium uptake. Standard final assay
solution conditions were as follows: 50 mM Tris, 5 mM MgCl2, 100 mM KCl, 10 mM oxalate, 68.5 µM CaCl2 total
Ca2+ (10 µM free Ca2+, 1.0-2.0
µCi of 45CaCl2), ± 1.25 mM ATP,
pH 6.8, at 37 °C.
The assay was initiated by adding 5 µg of protein from IM-enriched
fraction (HSP) to 1.5-ml tube containing the assay solution in a final
volume of 100 µl. The assay was terminated by filtration of assay
solution through Millipore filters presoaked in 0.25 M KCl
in a Millipore filtration manifold. The filters were washed with 250 mM sucrose, 40 mM NaCl at room temperature. The
filters were air-dried and dissolved with 1.0 ml of methyl Cellosolve (ethylene glycol monomethyl ether, Sigma, catalog no. E-5378). 10 ml of
UniversolTM brand scintillation fluid was added, and the
samples were counted by standard scintillation counting procedures. All
experiments were performed in triplicate.
Insulin Secretion and Content Assays--
Cells were seeded in
24-well plates at a cell density of 5 × 104
cells/well and cultured for 2 days as described above. For secretion experiments, the cells were pre-incubated for 30 min at 37 °C in 0.5 ml of KRB (115 mM NaCl, 24 mM
NaHCO3, 5 mM KCl, 1 mM
MgCl2, 2.5 mM CaCl2, 25 mM Hepes, pH 7.4) supplemented with 0.1% BSA. The cells
were then incubated for 120 min at 37 °C in 0.5 ml of KRB plus 0.1%
BSA containing 1% (v/v) vehicle dimethyl sulfoxide (Me2SO)
or 1 µM thapsigargin (Biomol) dissolved in
Me2SO at glucose concentrations of 0 and 15 mM.
After incubation, the assay solution was removed and cleared by
centrifugation at 15,000 × g, at 4 °C. The cells
remaining in each well were extracted with ice-cold acid ethanol. The
insulin concentrations of the assay solution and acid ethanol extracts,
diluted in KRB, were measured by insulin radioimmunoassay to determine
the insulin secretion and insulin content.
Statistical Analysis--
All values are expressed as mean plus
or minus S.E. Statistical analysis of ER Ca2+ uptake and
insulin secretion data was performed using two-way analysis of
variance. Differences with p values less than 0.01 were
considered significant.
 |
RESULTS |
IRS-1 and IRS-2 Localize to the Intracellular Membrane-enriched
Fractions in
-Cell Lines--
To determine IRS-1 localization in
-cells, subcellular fractions were prepared from the
TC6-F7
wild-type and IRS-1-overexpressing
-cell lines (
-IRS1).
Enrichment of ER in the HSP was confirmed by marker enzyme analysis.
Both
TC6-F7 and
-IRS1 HSP fractions showed enrichment in NADPH
cytochrome c reductase activity of 4.11 ± 1.09-fold
and 3.39 ± 0.54-fold, respectively (Table
I). The fractions from both cell lines
were then analyzed by quantitative Western blotting with an antibody
for IRS-1 and to determine the localization of this protein in
-cells. Immunoblotting studies show that IRS-1 distributes primarily
to the ER-enriched HSP fraction in the wild-type
TC6-F7 cell line
(2.2-fold enrichment compared with homogenate), whereas very little is
found in the cytosol (0.5-fold) (Fig. 1).
In the
-IRS1 cell line, IRS-1 also distributes predominantly to the
HSP (5.4-fold) with a small portion located in the cytosol (0.3-fold).
It has been previously reported that the
-IRS1 cell line expresses
2-fold more IRS-1 compared with the wild-type
TC6-F7 (7). A
comparison of the total IRS-1 protein in all fractions from both cell
lines shows that the
-IRS1 cell line contains 2.2-fold more IRS-1
than the
TC6-F7 cell line as expected. Interestingly, the additional
IRS-1 protein expressed in
-IRS1 cells does not distribute evenly to
the HSP and CYT fractions in the proportions determined for
TC6-F7
cells (2.2- and 0.5-fold, respectively). Instead, the additional IRS-1
protein appears to distribute only to the HSP, effectively increasing the total amount of IRS-1 protein on the ER and other cellular components found in the HSP fraction.

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Fig. 1.
IRS-1 and IRS-2 localization to the
IM-enriched fractions of -cell lines. IRS
protein distribution was performed by Western blot as described under
"Experimental Procedures." Fractions were prepared from TC6-F7
and -IRS1 cell lines. Fractions are as follows: homogenate, PM (PM,
nuclear material, cellular debris), SV, HSP (ER, Golgi, cytoskeleton),
and CYT. IRS-1 localizes to the HSP fraction in both TC6-F7 and
-IRS1 cell lines shown in upper blot. There is
scant IRS-1 present in the CYT cytosolic fraction in either cell line.
IRS-2 shows a similar distribution in both cell lines. GLUT2
distributes to the PM fraction (n = 3).
|
|
The distribution of IRS-2 in
-cell lines was determined by
immunoblotting. IRS-2 also distributes mostly to the HSP fraction in
the
TC6-F7 cell lines (2.6-fold), with a small portion located in
the cytosol (0.4-fold). The distribution of IRS-2 in
-IRS1 cells is
similar to wild-type cells with more IRS-2 in the HSP (1.8-fold)
compared with the cytosol (0.5-fold). These results are consistent with
previous findings (31-37). The fractions were also immunoblotted with
an anti-GLUT2 antibody to verify plasma membrane enrichment in the PM
fraction (Fig. 1). This analysis confirmed the enrichment of GLUT2 in
the PM fractions of both
-cell lines. Immunoblotting with
anti-calnexin antibody (data not shown) showed a similar pattern of ER
distribution. These data provide additional controls for fraction
enrichment and immunoblotting efficiency. Because SERCA by definition
is an ER protein, this result indicates that IRS proteins may be in the
same cellular compartment as SERCA.
Immunofluorescent Localization of IRS-1 and IRS-2 in
-Cell
Lines--
To get a clearer view of how IRS proteins distribute in
intact
-cells, the
TC6-F7 wild-type and
-IRS1 cell lines were
immunofluorescently labeled to determine whether IRS proteins
co-localize to the ER. The
-cell lines, grown on cover slips, were
incubated sequentially with different combinations of primary
antibodies (anti-IRS-1, anti-IRS-2, and anti-Bip/GRP78, an ER marker
protein) followed by anti-rabbit antibody labeled with cyanine2 (green)
and anti-mouse antibody labeled with Texas Red (red) as described under
"Experimental Procedures." The incubations were designed so that
every combination of primary and secondary antibodies (with the
exception of IRS-1 and IRS-2 together) was performed.
Images taken from cross-sections through immunofluorescently labeled
TC6 cells show the localization of IRS-1 in relation to the ER (Fig.
2, A-C). The green
fluorescent staining pattern of IRS-1 (Fig. 2A) is
consistent with the classical punctate staining pattern observed for
the ER, demonstrated by the red fluorescent staining for ER in the same
field (Fig. 2B). When these two images are superimposed
(Fig. 2C), the yellow fluorescent signal created by
overlapping of the green IRS-1 signal and red ER signal indicates that
IRS-1 co-localizes to the ER. In a similar fashion, the green fluorescent staining pattern of IRS-2 (Fig. 2D) closely
resembles the red fluorescent staining pattern of the ER (Fig.
2E). The presence of the overlapping yellow signal verifies
that IRS-2 also co-localizes to the ER (Fig. 2F). The
overlapping signal in these images is not the result of bleed-through
of signal from one fluorescent channel to the other. This was confirmed
by examining the images for each protein by itself in combination with
both immunofluorescently labeled secondary antibodies. For each
individual primary antibody, only the appropriate immunofluorescent
signal was detected (e.g. green only for IRS-1 alone) by
confocal microscopy (data not shown). In addition, incubation of the
cells with the fluorescently labeled secondary antibodies did not
contribute any background signal (Fig. 2, E and
F). Experiments performed in
-IRS1 cell lines show the
same localization of IRS proteins to the ER (data not shown). It should
be noted that the IRS protein and ER signals do not completely overlap.
We do not rule out the possibility that IRS proteins can localize to
other intracellular membranes or even the cytoskeleton (35). Indeed,
co-immunofluorescent labeling of the IRS proteins with GM130, a Golgi
marker, shows some overlap in signal (Fig.
3). It should be noted, however, that,
although the Golgi signal is localized to a smaller perinuclear area,
the IRS1/2 signal is much more extensive. A recent report has shown
that, in
-cells, portions of the ER interpolate between the
cisternae of the Golgi (42). The overlap in IRS protein signal with
Golgi could be caused by ER interpolation into the Golgi apparatus.

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Fig. 2.
IRS-1 and IRS-2 localization to the ER
of -cell lines by co-immunofluorescence.
IRS-1 localization was performed by Immunofluorescence as
described under "Experimental Procedures." IRS-1
(A-C) and IRS-2 (D-F) co-localize with
BiP/GRP78, an ER marker protein, in TC6-F7 cells and -IRS1 cells
(data not shown) (n = 3). A-C, cells were
incubated with anti-IRS-1 (1:100) and anti-BiP/GRP78 (1:50) together
for 1 h, followed by incubation with goat anti-rabbit-cy2 and
donkey anti-mouse-TR together for 1 h. D-F, for IRS-2
co-immunofluorescence, cells were incubated with anti-IRS-2 (1:100) and
anti-BiP/GRP78 (1:50) together for 1 h, followed by incubation
with goat anti-rabbit-cy2 and donkey anti-mouse-TR together for 1 h. Immunofluorescence data were obtained using confocal microscopy.
Data are representative of three experiments (n = 3).
Bkgd., background.
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Fig. 3.
IRS-1 and IRS-2 localization to the Golgi
of -cell lines by co-immunofluorescence.
IRS-1 localization was performed by immunofluorescence as described
under "Experimental Procedures." IRS-1 (A-C) and IRS-2
(D-F) co-localize with GM130, a Golgi marker protein, in
TC6-F7 cells and -IRS1 cells (data not shown) (n = 3). A-C, cells were incubated with anti-IRS-1 (1:100) and
anti-GM130 (1:50) together for 1 h, followed by incubation with
goat anti-rabbit-cy2 and donkey anti-mouse-TR together for 1 h.
D-F, for IRS-2 co-immunofluorescence, cells were
incubated with anti-IRS-2 (1:100) and anti-GM130 (1:50) together for
1 h, followed by incubation with goat anti-rabbit-cy2 and
donkey anti-mouse-TR together for 1 h. Immunofluorescence data
were obtained using confocal microscopy. Data are representative of
three experiments (n = 3). Bkgd.,
background.
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Taken together with the fractionation data, it appears that IRS
proteins are located primarily on the ER or other intracellular membranes in
-cells. This localization increases the likelihood that
IRS proteins are located in the vicinity of SERCA proteins.
Immuno-electron Microscopy Shows IRS-1 and SERCA3b Are Present in
ER-derived Microsomes--
To complement the previous studies,
microsomes in the HSP, prepared as described earlier, were examined by
electron microscopy after immunostaining for IRS-1, SERCA3b, and
BiP/GRP78. Because the microsomes in the HSP can be from either ER,
Golgi, or plasma membrane origin, confirmation of IRS-1 and SERCA3b
staining on ER-derived microsomes was necessary. Immunostaining for
IRS-1 concurrently with BiP/GRP78 shows that IRS-1 does indeed localize to ER-derived microsomes from the
TC6-F7 cell line (Fig.
4, A and D).
SERCA3b also co-localizes to ER-derived microsomes, as expected (Fig.
4, B and E). When these ER-derived microsomes are immunostained concurrently for IRS-1 and SERCA3b, we found staining for
both IRS-1 and SERCA3b on the same microsomes (Fig. 4, C and F). This same distribution is seen in immunostaining of
microsomes prepared from the
-IRS1 cell line (Fig.
5). These data confirm that IRS-1 not
only localizes to the intracellular membranes in
-cell lines, it
also localizes in part to the ER membrane where it is juxtaposed to
SERCA3b. The close proximity of these two proteins supports the
hypothesis that they may interact directly with one another.

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Fig. 4.
IRS-1 and SERCA3b proteins in ER-derived
microsomes of TC6-F7 cells. Localization
of IRS-1 and SERCA3b to ER-derived microsomes in TC6-F7 cell line
was performed as described under "Experimental Procedures." The
boxed sections on A-C are enlarged in
D-F. Grids adsorbed with a solution of HSP fraction were
incubated with the following combination of antibody solutions:
anti-IRS-1 (rabbit) + anti-BiP/GRP78 (mouse) (A and
D), anti-SERCA3b (rabbit) + anti-BiP/GRP78 (mouse)
(B and E), and anti-IRS-1 (mouse) + anti-SERCA3b
(rabbit) (C and F). All primary rabbit polyclonal
antibodies are labeled with 5-nm gold particles conjugated to
anti-rabbit secondary antibody, and all primary mouse monoclonal
antibodies are labeled with 18-nm gold particles conjugated to
anti-mouse secondary antibody. Immuno-gold-labeled proteins are
indicated by the arrows and are labeled as IRS-1
(i), SERCA3b (s), and BiP/GRP78 (e).
The size marker is for enlarged images D-F only. Images are
representative of four or five fields.
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Fig. 5.
IRS-1 and SERCA3b proteins in ER-derived
microsomes of -IRS1 cells. Localization
of IRS-1 and SERCA3b to ER-derived microsomes in -IRS1 cell line was
performed as described under "Experimental Procedures." The
boxed sections on A-C are enlarged in
D-F. Grids adsorbed with a solution of HSP fraction were
incubated with the following combination of antibody solutions:
anti-IRS-1 (rabbit) + anti-BiP/GRP78 (mouse) (A and
D), anti-SERCA3b (rabbit) + anti-BiP/GRP78 (mouse)
(B and E), and anti-IRS-1 (mouse) + anti-SERCA3b
(rabbit) (C and F). All primary rabbit polyclonal
antibodies are labeled with 5-nm gold particles conjugated to
anti-rabbit secondary antibody, and all primary mouse monoclonal
antibodies are labeled with 18-nm gold particles conjugated to
anti-mouse secondary antibody. Immuno-gold-labeled proteins are
indicated by the arrows and are labeled as IRS-1
(i), SERCA3b (s), and BiP/GRP78 (e).
The size marker is for enlarged images D-F only. Images are
representative of four or five fields.
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The number of gold particles labeling IRS-1 was determined for
every image taken from both
-cell lines (n = 9 for
each cell line). There were twice as many gold particles labeling IRS-1 in the
-IRS1 cell line images compared with the
TC6-F7 cell line
images, confirming the already reported level of overexpression (7). In
addition, ~70% of the IRS-1 staining vesicles are of ER origin
(71.1% for
TC6-F7 and 70.0% for
-IRS1 cell lines). These data
support our earlier studies showing IRS-1 in these
-cell lines is
located primarily on the ER.
IRS-1 Co-immunoprecipitates with SERCA3b in CHO-T and
-Cell
Lines--
To determine whether IRS-1 and SERCA3b proteins can
interact with one another in a cellular environment, lysates of CHO-T cells, either transfected with SERCA3b, a combination of SERCA3b and
IRS-1, or untransfected, were immunoprecipitated with polyclonal antibodies to IRS-1 and SERCA3b or normal rabbit serum. Each set of
transfected or untransfected CHO-T cells was treated with or without
insulin prior to lysis. Following immunoprecipitation, the samples were
immunoblotted with anti-IRS1 and anti-SERCA3b antibodies. Blotting for
IRS-1 shows that it is present in all of the IRS-1 immunoprecipitated
samples (Fig. 6, upper
blot). CHO-T cells contain endogenous IRS-1, the expression
of which is enhanced severalfold in the cell line transfected with
IRS-1. Insulin treatment does not affect the amount of protein
immunoprecipitated, but it does cause an upward shift in the
electrophoretic mobility of the IRS-1 band. This phenomenon, reported
by many others, reflects increased phosphorylation of amino acid
residues on the protein in response to insulin stimulation.
Immunoblotting of the SERCA3b-immunoprecipitated samples with
anti-SERCA3b shows a ~112-kDa band that is present only in the cell
lines transfected with SERCA3b; thus, there is no endogenous expression
of SERCA3b in CHO-T cells (Fig. 6, second blot
from top). Blotting of the IRS-1 immunoprecipitated samples with
-SERCA3b shows that SERCA3b co-immunoprecipitates with IRS-1 (Fig. 6, second blot from bottom). The
SERCA3b band detected in this blot is not the result of nonspecific
interactions because normal rabbit serum does not immunoprecipitate any
SERCA3b protein (Fig. 6, bottom blot). In
addition, this band is specifically immunoprecipitated by IRS-1 because
there is no SERCA3b protein detected in the untransfected cell lines,
which do contain endogenous IRS-1. The association of SERCA3b with
IRS-1 was enhanced with insulin stimulation by ~2-fold for cells
transfected with SERCA3b alone and ~6-fold for cells transfected with
both SERCA3b and IRS-1.

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Fig. 6.
IRS-1 co-immunoprecipitation with SERCA3b in
CHO-T cell lines. CHO-T cells were untransfected, transfected with
SERCA3b alone, or transfected with SERCA3b and IRS-1 for 24 h as
indicated under "Experimental Procedures." The cells were then
serum-starved for 3 h and treated acutely with or without 100 nM insulin for 5 min. Cell lysates were immunoprecipitated
(IP) with anti-IRS-1 (upper blot and
second blot from bottom), anti-SERCA3b
(second blot from top), or normal
rabbit serum (bottom blot) as indicated. Samples
were blotted with anti-IRS-1 (upper blot) and
anti-SERCA3b (three lower blots).
IRS-1 (IRS-1-labeled arrow) was detected as a
178-180-kDa band, and SERCA3b (S3b-labeled arrow) was
detected as a 112-114-kDa band by Western analysis. Data are
representative of three experiments (n = 3).
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TC6-F7 and
-IRS1 cell lines were immunoprecipitated and
immunoblotted in the same fashion as the transfected CHO-T cell lines.
Immunoblots of anti-IRS-1 and anti-SERCA3b immunoprecipitated
-cell
lysates show IRS-1 and SERCA3b are present in their respective immunoprecipitated samples (Fig. 7,
top blot and second blot
from top, respectively). Immunoblotting of anti-IRS-1
immunoprecipitated
-cell lysates with anti-SERCA3b antibody
demonstrates that SERCA3b can co-immunoprecipitate with IRS-1 in both
-cell lines (Fig. 7, second blot from
bottom). Unlike CHO-T cells, treatment of
-cells with
insulin did not significantly increase the amount of SERCA3b
co-immunoprecipitated with IRS-1 in the wild-type
TC6-F7 cell
lysate.

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Fig. 7.
IRS-1 co-immunoprecipitation with SERCA3b
in -cell lines. TC6-F7 and -IRS1
cell lines were cultured and treated as indicated under "Experimental
Procedures." The cells incubated in KRB for 30 min and treated
acutely with or without 100 nM insulin for 5 min. Cell
lysates were immunoprecipitated (IP) with anti-IRS-1
(upper blot and second blot
from bottom), anti-SERCA3b (second
blot from top), or normal rabbit serum
(bottom blot) as indicated. Samples were blotted
with anti-IRS-1 (upper blot) and anti-SERCA3b
(three lower blots). IRS-1
(IRS-1-labeled arrow) was detected as a 178-180-kDa band,
and SERCA3b (S3b-labeled arrow) was detected as a
112-114-kDa band by Western analysis. Data are representative of three
experiments (n = 3).
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ER Ca2+ Uptake in ER-enriched Fractions from
-Cell
Lines--
Previous work has shown that, in the IRS-1-overexpressing
-cell line,
-IRS1, ER calcium uptake through the ER
Ca2+ ATPase is decreased (7). To support the idea that this
effect is the result of a direct interaction between IRS-1 and SERCA rather than some defect in SERCA protein itself caused by
overexpressing IRS-1, we performed an ER 45Ca2+
uptake assay on microsomal fractions prepared as described earlier from
the
-cell lines.
Fig. 8 shows ER calcium uptake in
ER-enriched HSP fractions by
TC6-F7 and
-IRS1 cell lines. Both
cell lines show a time-dependent increase in ER calcium
uptake in the presence of ATP that is nearly identical (Fig.
8A). In the absence of ATP, the ER fractions from
TC6-F7
and
-IRS1 cells are not able to take up calcium. This confirms that
the ER calcium uptake measured in this experiment is through the
ATP-dependent SERCA pump. When the SERCA inhibitor thapsigargin is added to the assay, ER calcium uptake is inhibited in
both cell lines to a similar degree (Fig. 8B).
Ca2+ uptake decreases to 24.0 ± 4.7 nmol of
Ca2+/mg of protein in
TC6-F7 cells and 25.4 ± 3.8 nmol of Ca2+/mg of protein in
-IRS1 cells, representing
a 79 and a 79% decrease in Ca2+ uptake, respectively
(p = 0.0001). Thus, when SERCA protein is removed from
the intact cellular environment, it appears to function normally in
both cell lines. These data indicate that the decrease in ER calcium
uptake is not caused by a defect in SERCA protein itself. Rather, the
effect is more likely the result of protein interactions with IRS-1
that occur in intact cells when IRS-1 is activated and modulated by the
insulin receptor signaling pathway.

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Fig. 8.
ATP-dependent ER Ca2+
uptake in ER-enriched fractions from -cell
lines. A, Ca2+ uptake by ER in the HSP
fraction was measured as described under "Experimental Procedures."
There was no difference in ATP-dependent Ca2+
uptake between TC6-F7 wild-type cells (solid
square) and -IRS1 cells (solid
triangle). The absence of ATP resulted in minimal
Ca2+ uptake in both cell lines compared with blank
(open square and triangle). In the
absence of oxalate, Ca2+ uptake reached a maximum in less
than 10 min and then leveled off (closed diamond
and circle). The results for both cell lines without oxalate
were the same. Data shown are mean ± S.E. from three experiments
each in triplicate. B, 1 µM thapsigargin was
added at the beginning of the assay to inhibit ER Ca2+
uptake in TC6-F7 cells (open bar) and -IRS1
cells (closed bar). Data shown are mean ± S.E. from three experiments each performed in triplicate.
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Effect of Thapsigargin on Glucose-stimulated Insulin Secretion in
-Cell Lines--
Another previous finding with regard to cytosolic
calcium levels in
TC6-F7 and
-IRS1 cell lines showed that, when
wild-type
TC6-F7 cells were treated with thapsigargin, cytosolic
calcium levels increased to a level similar to that in the
-IRS1
cells. Because an increase in glucose-stimulated fractional insulin
secretion was also observed in
-IRS1 cells, it is possible that
thapsigargin treatment of wild-type
TC6-F7 cells could also increase
fractional insulin secretion to a similar level.
TC6-F7 cells
treated with 1 µM thapsigargin had no effect on
fractional insulin secretion in the absence of glucose (Fig.
9A). However, in the presence of 15 mM glucose, thapsigargin caused a 30% increase in
fractional insulin secretion, 0.182 ± 0.010, compared with
vehicle control, 0.140 ± 0.006 (p = 0.0010).
Furthermore, this increased level, roughly 20% secretion of total
insulin, is similar to the level seen in
-IRS1 cells (Fig.
9B). Thapsigargin treatment of
-IRS1 cells, even in the
presence of glucose, did not significantly change the level of
fractional insulin secretion. These data correlate with the effect on
cytosolic calcium we have previously reported and show that the
proposed effect on SERCA protein is indeed linked to insulin
secretion.

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Fig. 9.
Thapsigargin increases glucose-stimulated
fractional insulin secretion in wild-type TC6
cells, but not IRS1 cells.
Glucose-stimulated insulin secretion was performed as described under
"Experimental Procedures." A, -TC6 cells exposed
acutely to 1 µM thapsigargin showed a 30% increase in
fractional insulin secretion when stimulated by 15 mM
glucose, 0.182 ± 0.0103, compared with vehicle control,
0.140 ± 0.0064 (p = 0.0010). There was no
difference between conditions in -IRS1 cells. B,
fractional insulin secretion = insulin secretion (ng/ml)/insulin
content (ng/ml). All experiments were performed in triplicate
(n = 3).
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DISCUSSION |
In this study, we investigated whether IRS proteins could directly
interact with a
-cell SERCA isoform. We show that IRS-1 and IRS-2
localize to the endoplasmic reticulum of pancreatic
-cell lines and
that IRS-1 can directly interact with SERCA3b, one of the
-cell
SERCA isoforms. This interaction may be facilitated by the close
proximity of the two proteins on the ER. The interaction is enhanced
with insulin-stimulated phosphorylation of IRS-1.
Investigation into the subcellular localization of IRS-1 has generated
some confusion as to where this protein resides and the mechanism by
which it is activated in a particular location. Several early studies
examining the subcellular localization of IRS-1 by fractionation
demonstrated that IRS-1 is primarily localized to a fraction enriched
in intracellular membranes or low density microsomes, although IRS-1
was also detected in the cytosol (31-37). The authors of one study
proposed that parts of the intracellular membrane containing IRS-1 may
be juxtaposed to the plasma membrane facilitating activation of IRS-1
by the insulin receptor (32). More recent experiments that further
analyzed these fractions showed that a large portion of IRS-1 protein
present was insoluble when treated with detergent (35). IRS-2 also
showed this pattern of localization (37). The explanation provided for
this observation was that IRS-1 associates with large protein
complexes, like the cytoskeleton, which are resistant to solubilization
by detergents. These authors suggest that IRS-1 may be arrayed in a
cytoskeletal network just below the plasma membrane, which allows for
easy access to the insulin receptor. Another recent report has shown using indirect immunofluorescence that the pleckstrin homology and
phosphotyrosine binding domains of IRS-1 target the protein to the
plasma membrane (43). However, phosphorylation of IRS-1 appears to
occur before translocation to the plasma membrane.
In our study, we have employed three complementary techniques to show
that IRS-1 and IRS-2 co-localize to intracellular membranes in
pancreatic
-cell lines. Subcellular fractionation of the
TC6-F7 and the
-IRS1 cell lines showed that IRS proteins localized to the
intracellular membranes in the high speed pellet. Furthermore, overexpressed IRS-1 in the
-IRS1 cell line was primarily in the intracellular membrane fraction. Co-localization of IRS-1 and IRS-2
with the ER marker protein BiP/GRP78 was demonstrated by confocal
microscopy. Although the signals for the IRS proteins and ER marker
clearly overlapped, there was some additional immunofluorescent staining in other portions of the cell. Finally, immuno-electron microscopy experiments show that IRS-1 is present on ER-derived microsomal vesicles from
-cell lines, as confirmed by its
co-immunostaining with BiP/GRP78 and SERCA3b. Taken together, these
data present a strong case for IRS protein localization to
intracellular membranes, in particular the ER, in pancreatic
-cell
lines. The lack of complete localization of IRS-1 and IRS-2 to the ER
by confocal microscopy indicates that IRS proteins may distribute to
several pools within the cell Indeed, counting of IRS-1-labeled
vesicles in the immuno-electron microscopy study confirms that only
70% of the IRS-1-positive vesicles were of ER derivation. Considering the dynamic nature of insulin receptor signaling, it is possible that
IRS proteins translocate to several locations in the cell in response
to upstream signals. However, the majority of both phosphorylated and
non-phosphorylated IRS proteins are maintained on intracellular
membranes. IRS proteins in this pool can interact with other proteins
in the same location and potentially form large protein complexes that
are insoluble in detergents. An example of this comes from one study
that reports that IRS-1 can bind to the
3A subunit of the AP-3
adaptor complex resulting in intracellular membrane localization
(36).
The localization of IRS proteins to the ER increases the likelihood of
a direct interaction with
-cell SERCA isoforms. Because IRS proteins
have previously been shown to bind to several SERCA isoforms, including
a
-cell isoform SERCA2b (38), this interaction should occur in
-cell lines. We have demonstrated that the pancreatic
-cell SERCA
isoform, SERCA3b, co-immunoprecipitates with IRS-1 not only in both the
TC6-F7 and
-IRS1 cell lines, but also when expressed in a CHO-T
cell line. This interaction is enhanced when IRS-1 is phosphorylated by
insulin stimulation of the insulin receptor in the transfected CHO-T
cells. Overexpression of IRS-1 in this CHO-T system results in further
enhancement of IRS-1 binding to SERCA3b. This observation could explain
why IRS-1 overexpression in
-IRS1 cells inhibits SERCA function.
Because the overexpressed IRS-1 protein stays primarily in the ER,
there is a local increase in the concentration of IRS-1 that may favor
binding to SERCA. Increased binding of IRS-1 to SERCA could result in
the increased inhibition of SERCA and the increase in cytosolic calcium
we have previously reported (7). The lack of significant increase in the association of IRS-1 with SERCA3b in the
-cell lines could be a
result of our observation that IRS-1 rapidly dephosphorylates in our
-cell lysates unless phosphatase inhibitors are included in the
treatment (data not shown). In the presence of phosphatase inhibitors,
treated
-cell lysates can maintain IRS-1 in a phosphorylated form,
but the half-life of "activated" IRS-1 in the cell lysate has not
been determined. Given the labile nature of phosphorylated IRS-1 in the
-cell lysate, it is possible that during the immunoprecipitation protocol, some portion of the phosphorylated IRS-1 becomes
dephosphorylated. This would result in a reduced association of IRS-1
to SERCA3b approaching the level of untreated
-cells. These data do
demonstrate that IRS-1 and SERCA3b can co-immunoprecipitate in
-cells as well as transfected CHO-T cells.
Direct inhibition of SERCA by the pharmacological inhibitor
thapsigargin in a control
-cell line resulted in an increase in
cytosolic calcium concentration similar to that observed in the
-IRS1 cell line (7). Here we report that thapsigargin treatment of
TC6-F7 leads to an increase in fractional insulin secretion similar
to the increase seen in the IRS-1-overexpressing cell line.
Overexpression of IRS-1 alone is sufficient for SERCA inhibition
because no additional increase in fractional insulin secretion was
observed in the
-IRS1 cell line upon thapsigargin treatment.
Interestingly, thapsigargin only seems to increase fractional insulin
secretion when stimulatory levels of glucose are present. This may
indicate a glucose-dependent aspect to IRS-1 regulation of
cytosolic calcium and insulin secretion via SERCA inhibition.
In contrast to our findings, a recent study demonstrates that insulin
can cause
-cell hyperpolarization and diminish cytosolic calcium
oscillations in mouse islets incubated in 10 mM glucose via
an increase in whole-cell K+ conductance (44). This effect
could be mediated through a phosphatidylinositol 3-kinase-dependent pathway. However, in another study,
mouse islets incubated with the insulin-mimetic compound L-783,281
caused an increase in insulin release at 11 mM glucose
(45). There was no change in the pulsatile release of insulin under
these conditions. Treatment with the insulin mimetic at 3 mM glucose decreased the pulse frequency of insulin
release, but not the amount. Although Aspinwall et al. (24)
showed that insulin-induced insulin exocytosis could occur at 3 mM glucose, their experiments measured exocytosis events
and not actual insulin release, which may have been minimal at that
glucose concentration. Thus, although insulin can effect the
electrophysiology of the
-cell in a negative fashion, it can at the
same time increase insulin release. The effect of insulin appears to
depend on the amount of glucose present in the extracellular environment. It is also possible that the particular effect of insulin
in the
-cell depends on the cellular location of insulin signaling
components. This phenomenon is observed in other cell signaling pathways.
One potential caveat in the direct binding scheme proposed is that
overexpression of IRS-1 could have some effect on the production of
functional SERCA protein. If this is the case, then the resultant effects of IRS-1 overexpression could be caused by defects in SERCA
expression. Indeed, we have reported that, in the
-IRS1 cell line,
SERCA3b mRNA is decreased by 42%, but the functional consequences
of this are not clear (40). Here we demonstrate that ER calcium uptake
of SERCA in isolated microsomes is unaffected by the overexpression of
IRS-1. This may be the result of the fact that SERCA2b expression
remains unchanged and can compensate for the decrease in SERCA3b. The
observation that ER calcium uptake is decreased in whole
-IRS1 is
most likely a result of IRS-1 binding to both SERCA isoforms resulting
in inhibition. In this experiment, we are measuring SERCA activity in
the absence of any influence by IRS proteins, which accounts for this
discrepancy. Therefore, the phenomenon of IRS-1 inhibition of SERCA is
not the result of a defect in SERCA protein, but the direct binding to
and inhibition of SERCA by IRS-1.
We have previously described a novel positive feedback pathway in which
insulin can regulate insulin secretion in pancreatic
-cells (7).
This effect is mediated through a link between IRS-1 signaling and
modulation of intracellular Ca2+ by SERCA. This study
demonstrates that IRS-1 is present on ER membranes under basal
conditions and can directly bind to the
-cell isoforms of SERCA,
specifically SERCA3b. Insulin stimulation causes an increase in binding
of IRS-1 to SERCA3b, resulting in inhibition of the
Ca2+-ATPase, increased cytosolic Ca2+, and
increased fractional insulin secretion. However, this boost in
-cell
cytosolic Ca2+ via SERCA inhibition will only cause
increased insulin secretion in the presence of stimulatory levels of
glucose, providing evidence for another regulatory step in this process
that is glucose-dependent. One possibility is that SERCA
inhibition by IRS-1 lowers the threshold Ca2+ concentration
necessary for Ca2+-dependent insulin
exocytosis. In this scenario, smaller influxes of extracellular
Ca2+ could achieve insulin exocytosis, but glucose
stimulation would still be required to cause this influx. Another
possibility is that glucose or products of glucose oxidation may
regulate the interaction of IRS-1 and SERCA by some means yet to be
determined. In either case, this positive feedback pathway provides a
normal mechanism by which the
-cell can boost its response to high
levels of serum glucose.
Some recent studies suggest a strong role for SERCA3 in the development
of type 2 diabetes. An analysis of a type 2 diabetic patient population
showed that a unique subset of that population harbored specific
sequence variations that resulted in defective SERCA3 (46). Although
all the patients had a family history of diabetes, the genetic
inheritance of these mutations is not known. Further, SERCA3 mRNA
expression in islets from Goto-Kakizaki rats, a non-obese model for
type 2 diabetes, was significantly reduced (39). Our work shows that
SERCA3 mRNA levels are also decreased when IRS-1 is overexpressed
in
-cells (40). These data indicate that alterations in SERCA and
its link to insulin receptor signaling could lead to serious
malfunctions in
-cell homeostasis.