Insulin Receptor Substrate 1 Regulation of Sarco-endoplasmic Reticulum Calcium ATPase 3 in Insulin-secreting beta -Cells*

Prabhakar D. Borge Jr. and Bryan A. WolfDagger

From the Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received for publication, September 17, 2002, and in revised form, January 8, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have previously characterized an insulin receptor substrate 1 (IRS-1)-overexpressing beta -cell line. These beta -cells demonstrated elevated fractional insulin secretion and elevated cytosolic Ca2+ levels compared with wild-type and vector controls. This effect of IRS-1 may be mediated via an interaction with the sarco-endoplasmic reticulum calcium ATPase (SERCA). Here we demonstrate that IRS-1 and IRS-2 localize to an endoplasmic reticulum (ER)-enriched fraction in beta -cells using subcellular fractionation. We also observe co-localization of both IRS-1 and IRS-2 with ER marker proteins using immunofluorescent confocal microscopy. Furthermore, immuno-electron microscopy studies confirm that IRS-1 and SERCA3b localize to vesicles derived from the ER. In Chinese hamster ovary-T (CHO-T) cells transiently transfected with SERCA3b alone or together with IRS-1, SERCA3b co-immunoprecipitates with IRS-1. This interaction is enhanced with insulin treatment. SERCA3b also co-immunoprecipitates with IRS-1 in wild-type and IRS-1-overexpressing beta -cell lines. Ca2+ uptake in ER-enriched fractions prepared from wild-type and IRS-1-overexpressing cell lines shows no significant difference, indicating that the previously observed decrease in Ca2+ uptake by IRS-1-overexpressing cells is not the result of a defect in SERCA. Treatment of wild-type beta -cells with thapsigargin, an inhibitor of SERCA, resulted in an increase in glucose-stimulated fractional insulin secretion similar to that observed in IRS-1-overexpressing cells. The colocalization of IRS proteins and SERCA in the ER of beta -cells increases the likelihood that these proteins can interact with one another. Co-immunoprecipitation of IRS-1 and SERCA in CHO-T cells and beta -cells confirms that these proteins do indeed interact directly. Pharmacological inhibition of SERCA in beta -cells results in enhanced secretion of insulin. Taken together, our data suggest that interaction between IRS proteins and SERCA is an important regulatory step in insulin secretion.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The pancreatic beta -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 beta -cells (1-3). Glucose enters the beta -cell through GLUT2 transporters on the plasma membrane. Glucose metabolism begins with glucokinase, the beta -cell glucose sensor (4), and results in an increase in the intracellular ATP/ADP ratio. This causes closure of ATP-dependent K+ channels and beta -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 beta -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 beta -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 beta -cell and is involved in regulating key cellular processes (7-16). Insulin stimulation of the beta -cell IR results in tyrosine phosphorylation of the catalytic IR beta -subunit and IRS proteins (8). Mice with a pancreatic beta -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 beta -cell hyperplasia but no overt diabetic phenotype (17-20). Islets and beta -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 beta -cell mass, beta -cell failure, and overt type II diabetes (17, 22). Analysis of islets and beta -cells from IRS-2 knockout mice revealed no secretory defects, but altered beta -cell growth (23). This difference in phenotype of IRS knockouts indicates that these substrates mediate different functions in the beta -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 beta -cells and beta -cell lines found that IRS-1 also has an effect on calcium homeostasis. In one study, the overexpression of IRS-1 in the beta TC6-F7 cell line (beta -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 beta -cell line was treated with thapsigargin, a SERCA inhibitor, the cytosolic Ca2+ levels increased to the same level as the beta -IRS1 cell line. Furthermore, ER calcium uptake in a digitonin-permeabilized cell system was reduced in the beta -IRS1 cells compared with controls. In another study, insulin exocytosis and intracellular Ca2+ levels of single beta -cells from IRS-1-deficient mouse islets and IRS-1 null beta -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 beta -cell lines. More specifically, IRS-1 co-localizes with SERCA3b, one of two SERCA isoforms present in beta -cells, to endoplasmic reticulum-derived microsomes prepared from beta -cells. SERCA3b has also been shown to co-immunoprecipitate with IRS-1 in a model cell line and beta -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 beta -cells demonstrates that the beta -cell SERCA isoforms function normally and respond to inhibition with thapsigargin in the same fashion. Finally, treatment of wild-type beta -cells with thapsigargin resulted in an increase in glucose-stimulated fractional insulin secretion comparable with the increase observed for untreated IRS-1-overexpressing beta -cells. These results provide a functional link between the direct IRS-1/SERCA interaction and regulation of calcium homeostasis and insulin secretion.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines and Culture Media-- The beta -IRS1 cell line and clonal mouse beta -cell line beta 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 beta -Cell Lines-- Subcellular fractions were prepared using a modification of the method described by Colca et al. (41). Both the beta TC6-F7 and beta -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 beta -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 beta -Cells-- For immunofluorescence, beta -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 beta -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 beta TC6-F7 and beta -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). beta TC6-F7 and beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IRS-1 and IRS-2 Localize to the Intracellular Membrane-enriched Fractions in beta -Cell Lines-- To determine IRS-1 localization in beta -cells, subcellular fractions were prepared from the beta TC6-F7 wild-type and IRS-1-overexpressing beta -cell lines (beta -IRS1). Enrichment of ER in the HSP was confirmed by marker enzyme analysis. Both beta TC6-F7 and beta -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 beta -cells. Immunoblotting studies show that IRS-1 distributes primarily to the ER-enriched HSP fraction in the wild-type beta 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 beta -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 beta -IRS1 cell line expresses 2-fold more IRS-1 compared with the wild-type beta TC6-F7 (7). A comparison of the total IRS-1 protein in all fractions from both cell lines shows that the beta -IRS1 cell line contains 2.2-fold more IRS-1 than the beta TC6-F7 cell line as expected. Interestingly, the additional IRS-1 protein expressed in beta -IRS1 cells does not distribute evenly to the HSP and CYT fractions in the proportions determined for beta 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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Table I
NAPDH cytochrome c reductase activity (-fold enrichment) in beta -cell fractions
*, p < 0.005.


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Fig. 1.   IRS-1 and IRS-2 localization to the IM-enriched fractions of beta -cell lines. IRS protein distribution was performed by Western blot as described under "Experimental Procedures." Fractions were prepared from beta TC6-F7 and beta -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 beta TC6-F7 and beta -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 beta -cell lines was determined by immunoblotting. IRS-2 also distributes mostly to the HSP fraction in the beta TC6-F7 cell lines (2.6-fold), with a small portion located in the cytosol (0.4-fold). The distribution of IRS-2 in beta -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 beta -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 beta -Cell Lines-- To get a clearer view of how IRS proteins distribute in intact beta -cells, the beta TC6-F7 wild-type and beta -IRS1 cell lines were immunofluorescently labeled to determine whether IRS proteins co-localize to the ER. The beta -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 beta 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 beta -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 beta -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 beta -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 beta TC6-F7 cells and beta -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 beta -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 beta TC6-F7 cells and beta -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.

Taken together with the fractionation data, it appears that IRS proteins are located primarily on the ER or other intracellular membranes in beta -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 beta 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 beta -IRS1 cell line (Fig. 5). These data confirm that IRS-1 not only localizes to the intracellular membranes in beta -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 beta TC6-F7 cells. Localization of IRS-1 and SERCA3b to ER-derived microsomes in beta 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 beta -IRS1 cells. Localization of IRS-1 and SERCA3b to ER-derived microsomes in beta -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.

The number of gold particles labeling IRS-1 was determined for every image taken from both beta -cell lines (n = 9 for each cell line). There were twice as many gold particles labeling IRS-1 in the beta -IRS1 cell line images compared with the beta 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 beta TC6-F7 and 70.0% for beta -IRS1 cell lines). These data support our earlier studies showing IRS-1 in these beta -cell lines is located primarily on the ER.

IRS-1 Co-immunoprecipitates with SERCA3b in CHO-T and beta -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 alpha -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).

beta TC6-F7 and beta -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 beta -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 beta -cell lysates with anti-SERCA3b antibody demonstrates that SERCA3b can co-immunoprecipitate with IRS-1 in both beta -cell lines (Fig. 7, second blot from bottom). Unlike CHO-T cells, treatment of beta -cells with insulin did not significantly increase the amount of SERCA3b co-immunoprecipitated with IRS-1 in the wild-type beta TC6-F7 cell lysate.


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Fig. 7.   IRS-1 co-immunoprecipitation with SERCA3b in beta -cell lines. beta TC6-F7 and beta -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).

ER Ca2+ Uptake in ER-enriched Fractions from beta -Cell Lines-- Previous work has shown that, in the IRS-1-overexpressing beta -cell line, beta -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 beta -cell lines.

Fig. 8 shows ER calcium uptake in ER-enriched HSP fractions by beta TC6-F7 and beta -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 beta TC6-F7 and beta -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 beta TC6-F7 cells and 25.4 ± 3.8 nmol of Ca2+/mg of protein in beta -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 beta -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 beta TC6-F7 wild-type cells (solid square) and beta -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 beta TC6-F7 cells (open bar) and beta -IRS1 cells (closed bar). Data shown are mean ± S.E. from three experiments each performed in triplicate.

Effect of Thapsigargin on Glucose-stimulated Insulin Secretion in beta -Cell Lines-- Another previous finding with regard to cytosolic calcium levels in beta TC6-F7 and beta -IRS1 cell lines showed that, when wild-type beta TC6-F7 cells were treated with thapsigargin, cytosolic calcium levels increased to a level similar to that in the beta -IRS1 cells. Because an increase in glucose-stimulated fractional insulin secretion was also observed in beta -IRS1 cells, it is possible that thapsigargin treatment of wild-type beta TC6-F7 cells could also increase fractional insulin secretion to a similar level. beta 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 beta -IRS1 cells (Fig. 9B). Thapsigargin treatment of beta -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 beta TC6 cells, but not beta IRS1 cells. Glucose-stimulated insulin secretion was performed as described under "Experimental Procedures." A, beta -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 beta -IRS1 cells. B, fractional insulin secretion = insulin secretion (ng/ml)/insulin content (ng/ml). All experiments were performed in triplicate (n = 3).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we investigated whether IRS proteins could directly interact with a beta -cell SERCA isoform. We show that IRS-1 and IRS-2 localize to the endoplasmic reticulum of pancreatic beta -cell lines and that IRS-1 can directly interact with SERCA3b, one of the beta -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 beta -cell lines. Subcellular fractionation of the beta TC6-F7 and the beta -IRS1 cell lines showed that IRS proteins localized to the intracellular membranes in the high speed pellet. Furthermore, overexpressed IRS-1 in the beta -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 beta -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 beta -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 sigma 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 beta -cell SERCA isoforms. Because IRS proteins have previously been shown to bind to several SERCA isoforms, including a beta -cell isoform SERCA2b (38), this interaction should occur in beta -cell lines. We have demonstrated that the pancreatic beta -cell SERCA isoform, SERCA3b, co-immunoprecipitates with IRS-1 not only in both the beta TC6-F7 and beta -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 beta -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 beta -cell lines could be a result of our observation that IRS-1 rapidly dephosphorylates in our beta -cell lysates unless phosphatase inhibitors are included in the treatment (data not shown). In the presence of phosphatase inhibitors, treated beta -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 beta -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 beta -cells. These data do demonstrate that IRS-1 and SERCA3b can co-immunoprecipitate in beta -cells as well as transfected CHO-T cells.

Direct inhibition of SERCA by the pharmacological inhibitor thapsigargin in a control beta -cell line resulted in an increase in cytosolic calcium concentration similar to that observed in the beta -IRS1 cell line (7). Here we report that thapsigargin treatment of beta 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 beta -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 beta -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 beta -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 beta -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 beta -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 beta -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 beta -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 beta -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 beta -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 beta -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 beta -cells (40). These data indicate that alterations in SERCA and its link to insulin receptor signaling could lead to serious malfunctions in beta -cell homeostasis.

    ACKNOWLEDGEMENTS

We thank the following University of Pennsylvania Diabetes Center Core facilities: the Insulin RIA Core for analysis of insulin secretion samples and the Biomedical Imaging Core for use of the confocal microscope and assistance with all electron microscopy studies.

    FOOTNOTES

* This work was supported by grants from the American Diabetes Association (to P. D. B. and B. A. W.) and by National Institutes of Health (NIH) Grant DK49814 (to B. A. W.). The Radioimmunoassay Core and Biomedical Imaging Core of the Penn Diabetes Center are supported by NIH Grant DK19525.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, 5135 Main, 34th St. and Civic Center Blvd., Philadelphia, PA 19104-4399. Tel.: 215-590-2869; Fax: 215-590-1021; E-mail: wolfb@mail.med.upenn.edu.

Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M209521200

    ABBREVIATIONS

The abbreviations used are: IR, insulin receptor; IRS, insulin receptor substrate(s); SERCA, sarco-endoplasmic reticulum calcium ATPase; ER, endoplasmic reticulum; IM, intracellular membrane; HSP, high speed pellet; DMEM, Dulbecco's modified Eagle's medium; PM, plasma membrane pellet; SV, secretory vesicle and mitochondria pellet; CYT, cytosol; KRB, Krebs-Ringer buffer; TR, Texas Red; cy2, cyanine 2; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TBS-T, Tris-buffered saline with Tween 20; FBS, fetal bovine serum; MES, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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