Insulin Receptor Substrate 1-induced Inhibition of Endoplasmic Reticulum Ca2+ Uptake in beta -Cells
AUTOCRINE REGULATION OF INTRACELLULAR Ca2+ HOMEOSTASIS AND INSULIN SECRETION*

Gang G. Xu, Zhi-yong Gao, Prabhakar D. Borge Jr., and Bryan A. WolfDagger

From the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To understand the role of the insulin receptor pathway in beta -cell function, we have generated stable beta -cells (beta IRS1-A) that overexpress by 2-fold the insulin receptor substrate-1 (IRS-1) and compared them to vector-expressing controls. IRS-1 overexpression dramatically increased basal cytosolic Ca2+ levels from 81 to 278 nM, but it did not affect Ca2+ response to glucose. Overexpression of the insulin receptor also caused an increase in cytosolic Ca2+. Increased cytosolic Ca2+ was due to inhibition of Ca2+ uptake by the endoplasmic reticulum, because endoplasmic reticulum Ca2+ uptake and content were reduced in beta IRS1-A cells. Fractional insulin secretion was significantly increased 2-fold, and there was a decrease in beta IRS1-A insulin content and insulin biosynthesis. Steady-state insulin mRNA levels and glucose-stimulated ATP were unchanged. High IRS-1 levels also reduced beta -cell proliferation. These data demonstrate a direct link between the insulin receptor signaling pathway and the Ca2+-dependent pathways regulating insulin secretion of beta -cells. We postulate that during regulated insulin secretion, released insulin binds the beta -cell insulin receptor and activates IRS-1, thus further increasing cytosolic Ca2+ by reducing Ca2+ uptake. We suggest the existence of a novel pathway of autocrine regulation of intracellular Ca2+ homeostasis and insulin secretion in the beta -cell of the endocrine pancreas.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The insulin-secreting beta -cell of the endocrine pancreas has a central role in regulating glucose homeostasis (1, 2). It is now recognized that beta -cell failure is a major contributing factor to type 2 diabetes mellitus, thus emphasizing the importance of elucidating the normal mechanisms of insulin secretion (3, 4). Glucose oxidation by the beta -cell is essential for insulin secretion. In particular, glucokinase, the first step in glycolysis, has been convincingly shown to be the beta -cell glucose sensor (5). beta -Cell metabolism of glucose results in an increase in the ATP/ADP ratio leading to closure of the KATP channel, depolarization of the beta -cell, and influx of extracellular Ca2+ through voltage-dependent Ca2+ channels. The subsequent increase in intracellular Ca2+ then activates insulin exocytosis. The possibility of other signaling pathways involved in glucose-induced insulin secretion has also been suggested (6-11).

Since the discovery of the insulin receptor in insulin-secreting beta -cells by Rothenberg and colleagues (12, 13), a rapidly growing body of evidence indicates that the insulin receptor signaling pathway is active in pancreatic beta -cells (14) and plays an important role in beta -cell regulation (4, 12-17). Activation of the beta -cell insulin receptor (IR)1 results in rapid tyrosine phosphorylation of the IR beta -subunit and the IR substrate proteins (12). Deletion of IR results in neonatal death in mice (18, 19) and leprechaunism in humans (20). Mice with heterozygous null alleles of IR and insulin receptor substrate 1 (IRS-1) (IR/IRS-1+/-) exhibit hyperinsulinemia and beta -cell hyperplasia and develop overt diabetes (21). Knockouts of the IRS-1 and IRS-2 produce different effects. Inactivation of IRS-1 (IRS-1-/-) leads to mild insulin resistance, hyperinsulinemia, and beta -cell hyperplasia with no overt diabetes syndrome (4, 17, 22). In contrast, inactivation of IRS-2 (IRS-2-/-) results in beta -cell failure and causes type 2 diabetes (17). The differential effects of IRS-1 and IRS-2 knockout indicate that the two major IR substrates mediate differential signals in beta -cells, but the mechanisms accounting for such differential regulation and for IRS-1 function are still unknown.

Cellular Ca2+ is a critical element in beta -cell regulation. Rising intracellular Ca2+ ([Ca2+]i) is an obligated step for glucose induced insulin secretion (1, 23). Abnormal [Ca2+]i is a common defect in both insulin-dependent type 1 diabetes and insulin-independent type 2 diabetes (24). Altered Ca2+ metabolism has also been reported to affect beta -cell function including insulin biosynthesis (25, 26). The endoplasmic reticulum (ER) plays an important role in the regulation of intracellular Ca2+ concentrations (27-29). Endoplasmic reticulum Ca2+-ATPase (SERCA) is the major calcium pump that sequestrates cytosol Ca2+ into ER lumen (28, 30). Thapsigargin, a nonphorboid tumor promoter, specifically inhibits ER Ca2+-ATPase activity (31). Addition of thapsigargin to pancreatic beta -cells leads to elevated cytosol Ca2+ concentration and enhanced short term glucose-stimulated insulin secretion (32). Recent data showed that IRSs may directly interact with ER Ca2+-ATPase (SERCA1 and SERCA2) in a tyrosine phosphorylation-dependent manner in muscle and heart (33). This finding suggests that insulin may via insulin receptor signaling pathway regulate ER Ca2+-ATPase activity, therefore influencing cellular Ca2+ homeostasis. It is currently unknown whether insulin exerts any regulatory role in beta -cell Ca2+ homeostasis.

To dissect the role of IRS-1 in beta -cell function, we have overexpressed IRS-1 in an insulin-secreting beta -cell line. We show that IRS-1 regulates beta -cell Ca2+ homeostasis, insulin biosynthesis, and beta -cell proliferation and that elevated expression of IRS-1 induces abnormal Ca2+ homeostasis and beta -cell dysfunction.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Lines and Culture Media-- The clonal mouse beta -cell line beta TC6-F7 and culture conditions were previously described (15, 34). In brief, cells were maintained in high glucose Dulbecco's modified Eagle's medium (25 mM glucose; Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, 50 µg/ml streptomycin and incubated at 37 °C in a 10% CO2/90% air humidified incubator. The Chinese hamster ovary-T cell line was a kind gift from Dr. R. Roth (Stanford University School of Medicine, Stanford, CA) and was maintained in F-12 medium with 5% fetal bovine serum, 100 units/ml penicillin, 50 µg/ml streptomycin at 37 °C in a 5% CO2/95% air atmosphere.

Construction of the IRS-1 Expression Plasmid-- The IRS-1 expression vector was constructed as follows. A 3.9-kilobase DNA fragment containing the mouse IRS-1 cDNA and the c-Myc epitope tag was excised from pQE31-mIRS1 (kindly provided by Dr. Ronald Kahn, Joslin Diabetes Center, Boston, MA) with EcoICRI and HindIII. The fragment was blunt-ended with Klenow fragment (New England Biolabs, Beverly, MA) and ligated into a SmaI digested vector pCI-Neo (Promega, Madson, WI). The resulting plasmid pCMV-IRS1 was purified with QIAGEN Maxi-purification Kit (Qiagen, Chatsworth, CA). Enzymes for recombinant DNA procedures were from Promega or New England Biolabs.

Transfection of beta -Cells-- Cells were transfected with cationic liposome reagent DMRIE-C (Life Technologies, Inc.) as described before (15). The transfected cells were selected with 500 µg/ml neomycin (Geneticin, Life Technologies, Inc.) for 4 weeks, and the surviving colonies (defined as passage 4) were individually picked and transferred to 24-well plates. Then the IRS-1 and c-Myc protein levels were determined with anti-IRS1 polyclonal antibody (catalog no. 06-248, Upstate Biotechnology, Lake Placid, NY) and anti-c-Myc monoclonal antibody 9E10 (catalog no. sc-40, Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Immunoprecipitation and Western Analysis-- Preparation of cell lysates, immunoprecipitation, and Western blotting were performed essentially as described previously (12, 35). Tyrosine-phosphorylated proteins were detected with rabbit polyclonal anti-phosphotyrosine antibody K-18 (kindly provided by Dr. P. Rothenberg, University of Pennsylvania). A secondary antibody, rabbit anti mouse IgG (Sigma, catalog no. M7023), was used for c-Myc antibody immunoprecipitation and immunoblotting.

Insulin Assay and beta -Cell Metabolism (MTT) Assay-- Insulin content and secretion assays were performed essentially as described before (12, 35). The metabolic rate of the beta -cells was indirectly measured by the production of formazan, which is produced from 3-(4, 5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) as the cells metabolize glucose. The MTT assay was performed essentially as described (36, 37). In brief, the cells were preincubated in Krebs-Ringer buffer (KRB) (115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 25 mM glucose, and 25 mM Hepes, pH 7.4) for 30 min. Then, MTT was added to a final concentration of 0.5 mg/ml. Incubation was continued for another 60 min at 37 °C (5% CO2/95% air). The supernatant was removed, and 500 µl of 2-propanol were added to dissolve the formazan. The optical density of the solution was measured at 562 nm in a Microkinetics Plate Reader (Fisher).

Measurement of Cellular Nucleotides-- Cellular contents of nucleotides including ATP were measured by high performance liquid chromatography (HPLC) as described before (38). In brief, cells were washed with ice-cold phosphate-buffered saline and extracted with 1 ml of 5% trichloracetic acid. Cells were scraped, transferred to 1.5-ml Eppendorf tubes, incubated 5 min at +4 °C, and centrifuged at 14,000 × g (10 min at +4 °C). The supernatant was collected into borosilicate 12 × 75-mm tubes, extracted three times with 1 ml of diethyl ether, and centrifuged, and the ether phase was discarded. Samples (300 µl) and known amounts of standard NAD, ADP, GDP, ATP, GTP, UTP, CTP, and TTP were analyzed on a Varian HPLC apparatus (Varian, Sugarland, Texas), using a 250 × 4.6 mm Partisil SAX (10 µm) column equipped with a guard column. The gradient of solvent A (1.25 MNaH2PO4, pH 3.8) over 80 min was as follows: 0 min, 100% water; 1-15 min, 1% A; 25 min, 28% A; 45 min, 32% A; 70 min, 50% A; 70-80 min, 100% A; 80-95 min, 100% water. The flow rate was 1.5 ml/min. Nucleotides were detected by UV spectrophotometry (340 nm) and quantitated by comparison to the corresponding standard curve run in parallel. The typical retention times were as follows : 8 min for NAD, 10 min for AMP, 35 min for ADP, 27 min for GDP, 32 min for UTP, 34.5 min for CTP, 34.8 min for TTP, 40 min for ATP, and 46 min for GTP.

Cytosolic free Ca2+ Measurement-- For cytosolic free Ca2+ measurement, the cells were seeded on microscope glass coverslips (Fisher) coated with poly-D-lysine (Sigma), and grown for 2 days. Cells were then loaded with the calcium indicator fura-2 for 30 min at 37 °C in 2 ml of KRB plus 0.1% BSA and supplemented with 2.0 µM fura-2 acetoxymethylester (Molecular Probes, Eugene, OR) and 0.2 mg/ml pluronic F-127 (Molecular Probes). The coverslip with the loaded cells was then mounted in a perifusion chamber placed on the homeothermic platform of an inverted Zeiss microscope. The cells were superfused with Krebs-Ringer buffer (0.1% BSA) at 37 °C at a flow rate of 1.5 ml/min. For experiments using KRB without Ca2+, CaCl2 was omitted and 1 mM EGTA (Sigma) was added to chelate all Ca2+. Ca2+ measurement is as follows and was also described in detail elsewhere (37, 39). The microscope was used with a × 40 oil objective. Fura-2 was successively excited at 334 and 380 nm by means of two narrow band-pass filters. The emitted fluorescence was filtered through a 520 nm filter, captured with an Attofluor CCD video camera at a resolution of 512 × 480 pixels, digitized into 256 gray levels and analyzed with version 6.00 of the Attofluor RatioVision software (Atto Instruments, Rockville, MD). The concentration of Ca2+ at each pixel was calculated by comparing the ratio of fluorescence to an in vitro two-point calibration curve. The Ca2+ concentration is presented by averaging the values of all pixels of a cell body. Data were collected from at least 10 individual cells in each measurement at an interval of 4.5 s.

The endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin was purchased from Biomol (Plymouth Meeting, PA) and dissolved in dimethyl sulfoxide.

Measurement of Endoplasmic Reticulum Ca2+ Uptake-- Endoplasmic reticulum Ca2+ uptake was measured as described (40, 41). Briefly, the cells were trypsinized and washed twice with KRB without Ca2+ or glucose (0.1% BSA). Then, they were permeabilized with 20 µg/ml digitonin (Sigma) in KRB without Ca2+ or glucose (0.1% BSA) at 37 °C for 10 min. The cells were then incubated for 30 min in TES buffer (100 mM TES, pH 7.2, 100 mM KCl, 2.5 mM MgCl2, 0.2 mM EGTA, 5 µCi/ml of 45Ca (ICN, Lisle, IL), 5 µCi/ml of 3H2O (ICN) for diffusible space correction, 5 µg/ml of ruthenium red, 0.1% BSA) at the indicated Ca2+ concentration. The incubation medium was removed by aspiration, and the cells were solubilized with 100 µl of 1 M NaOH and neutralized with 100 µl of 1 M HCl. Radioactivity content of the endoplasmic reticulum was measured by liquid scintillation spectrometry. Calcium concentrations were titrated with a calcium electrode (Orion, Beverly, MA). Calculation of endoplasmic reticulum calcium uptake was as described before (41).

Pulse-Chase Labeling of beta -Cells and Determination of Insulin Biosynthesis-- The rate of insulin biosynthesis was determined with [3H]leucine pulse-chase labeling assay. The cells were seeded in 6-well culture dishes at 3 × 105 cells/well and grown for 2 days in high glucose Dulbecco's modified Eagle's medium (Life Technologies, Inc.). The cells were then washed twice with KRB buffer and labeled with 40 µCi/ml of L-[3,4,5-3H(N)]leucine (6660 GBq/mmol, NEN Life Science Products) in KRB buffer supplemented with 0.25% BSA and 0 or 16 mM glucose. After being labeled for 30 min, the cells were chased with 1 mM cold leucine for 30 min at the same glucose concentration as used in the labeling process. The cells were then extracted with 1 M acetic acid and sonicated. One hundred µl of the clarified acetic acid extract were used for trichloroacetic acid precipitation as described (15) to determine the total cellular content of 3H-labeled proteins. The 3H-labeled insulin immunoreactive material was immunoprecipitated with 3 µl of undiluted guinea pig anti-bovine insulin serum (ICN) and protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech). The radioactivity was determined by liquid scintillation counting. Normal guinea pig serum (Linco, St. Charles, MO) was used as background control. Recovery of insulin immunoreactive material was more than 90% under the assay condition as determined with 125I-labeled insulin.

RNA Protection Assay-- RNA protection assays were performed according to Ambion's protocol (Ambion, Austin, TX). Briefly, the cells were grown in culture medium for 2 days and then trypsinized and washed twice with Dulbecco's phosphate-buffered saline. To 4 × 106 cells, 0.4 ml of Ambion's lysis/denaturation solution (direct protect kit, Ambion) were added to lyse the cells. An insulin probe was generated by in vitro transcription using pGEM-rPPI (gift of Dr. Christopher Rhodes, Joslin Diabetes Center, Boston, MA) as a template. For internal control, a probe for 18 S RNA was obtained by in vitro transcription using pT7RNA18S (Ambion) as a template. Labeling of the 18 S RNA probe was done with [32P]UTP in the presence of 500 µM cold UTP to lower the specific activity. The labeled transcripts were gel purified as described in Ambion's manual. The insulin probe was 430 bases in length and completely complementary to the insulin mRNA. The 18 S rRNA probe was 116 nucleotides in length, 80 of which are complementary to 18 S rRNA (Ambion). Hybridization, RNase and protease digestion, and separation detection were carried out as per Ambion's instructions. The results were quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Data Analysis-- Data were analyzed by paired t test or one-way or two-way ANOVA; p <=  0.05 was considered significant. Nonlinear regression and curve fitting were performed with PRISM 2.01 (GraphPad Software, San Diego, CA).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Overexpression of IRS-1 in beta -Cells-- We overexpressed IRS-1 in a clonal beta -cell line beta TC6-F7. The exogenous IRS-1 had a c-Myc tag at its C terminus and migrated slightly slower than the endogenous IRS-1 in SDS-gels (Fig. 1A, lanes 1, 3, and 4). beta -IRS1-A, one of the 11 stable transfectants tested, expressed IRS-1 protein two times (199 ± 36%) more than the controls (p = 0.02) (Fig. 1B). Fifty percent of the increase was contributed by the exogenous IRS1-Myc; the other 50% was from the endogenous IRS-1. Addition of 100 nM insulin led to rapid tyrosine phosphorylation of proteins in the 160-180 kDa range that co-migrated with the IRS-1 protein (Fig. 1C, upper gel, lanes 2, 4, and 6) (also see Ref. 12). A 120-kDa protein (p120) was also heavily tyrosine-phosphorylated in the beta -cells. The abundance of p120 and its extent of tyrosine phosphorylation are relatively stable in the three beta -cell lines (Fig. 1C and data not shown), and it was therefore used to normalize the level of IRS-1 phosphorylation. Normalized level of IRS-1 tyrosine phosphorylation in the beta IRS1-A cells was 2-fold higher than that in the control cells (Fig. 1, C, upper gel, lane 4, and D). This is proportional to the elevated IRS-1 protein level in the beta IRS1-A cells. These data demonstrated that the excess IRS-1 was tyrosine-phosphorylated to the same extent as the endogenous substrate upon insulin stimulation.


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Fig. 1.   Western analysis of the IRS-1 protein and tyrosine phosphorylation of IRS-1. A, Western analysis. Cells were lysed as described under "Materials and Methods." For c-Myc epitope detection (lanes 1 and 2), 250 µg of total protein were immunoprecipitated and blotted with c-Myc antibody 9E10. For IRS-1 detection (lanes 3-6), 16 µg of total protein were loaded in duplicate and blotted with IRS-1 antibody. Positions of IRS1-Myc and endogenous IRS-1 are indicated to the right. Eleven clones were analyzed. Data shown are from a representative clone beta IRS1-A. B, quantitation of IRS-1 protein. IRS-1 protein levels by immunoblots were quantitated with a PhosphorImager and expressed as a percentage of the IRS-1 level in parental beta TC6-F7 cells. Data are shown as mean ± S.E., n = 6, from three independent experiments. *, p = 0.02. C, tyrosine phosphorylation. Cells were treated with or without insulin (15). Twenty µg of total protein were loaded onto SDS gels for phosphotyrosine and IRS-1 Western analysis. Anti-phosphotyrosine (alpha -PY) detection is shown in the upper panel. The IRS-1 protein was detected in duplicate blots with alpha -IRS1 antibody and is shown in the lower panel. Chinese hamster ovary-T is a cell line overexpressing the insulin receptor and is used as positive control (lanes 7 and 8). D, quantitation of IRS-1 tyrosine phosphorylation. Radioactivity of the protein bands was quantitated with a PhosphorImager. The IRS-1 signal was normalized to the p120 band (IRS1/p120) to correct for small sample loading variation. Data were collected from two experiments with n = 6 and are shown as mean ± S.E. **, p = 0.001. Cell lines were as follows: beta IRS1-A, stable transfectant expressing the tagged IRS1-Myc protein; beta TC6-F7, parental cell; NEO, beta TC6-F7 cells transfected with vector only.

Down-regulation of Insulin Content and Secretion by Overexpression of IRS-1-- Nine of the transfected clones that showed detectable c-Myc signal and increased IRS-1 levels, including beta IRS1-A, were tested for their insulin content. They all exhibited lowered insulin content. Data from the beta IRS1-A clone are shown in Fig. 2. The beta IRS1-A cells had significantly lowered insulin content: 29.0 ± 2.9 ng/105 cells versus 114 ± 2.2 ng/105 cells (Neo control), p = 0.0001 (Fig. 2A). Net insulin secretion was also reduced 61% at 0 mM glucose (G0) and 58% at 15 mM glucose (G15) (Fig. 2B). Insulin secretion of both beta IRS1-A and control cell lines was glucose- and extracellular calcium-dependent. Removal of extracellular Ca2+ and addition of 1 mM EGTA completely abolished glucose-stimulated insulin secretion. Addition of either the phosphatidylinositol 3-kinase inhibitor wortmannin (100 nM) or the p70 ribosomal S6 protein kinase inhibitor rapamycin (100 nM) did not change insulin content and secretion of beta IRS1-A cells (data not shown).


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Fig. 2.   Insulin contents and glucose-stimulated insulin secretion. Insulin contents and secretion were assayed as described under "Materials and Methods." A, insulin content; B, glucose-stimulated insulin secretion. Open bars, 0 mM glucose; filled bars, 15 mM glucose. All data represent means ± S.E. from at least two independent experiments each performed in triplicate. Cell number was determined by cell counting and measurement of cellular DNA content. **, p = 0.0001. C, fractional insulin secretion. Secreted insulin at each glucose concentration was normalized with total cellular insulin content (secreted insulin/total insulin) and expressed as a percentage. The difference between the two fractional secretion curves is significant as analyzed with the ANOVA test (p < 0.05).

Although the net amount of insulin secreted by the IRS-1-overproducing cells was reduced as mentioned above, glucose-stimulated fractional insulin secretion (the ratio of secreted insulin/total insulin content, expressed as a percentage) was significantly increased in beta IRS1-A cells (Fig. 2C). This increased fractional insulin secretion was glucose-dependent. At 0 mM glucose, fractional insulin secretion of the beta IRS1-A cells (4.3 ± 0.9%) was not significantly different from that of the Neo control (3.0 ± 0.6%) (p > 0.5). At glucose concentrations above 1 mM (stimulatory glucose concentrations for this beta -cell line), fractional insulin secretions were increased more than 2-fold compared with the Neo control (Fig. 2C) (p < 0.04). These data indicate that overexpression of IRS-1 may enhance the capacity of the beta -cell to secrete insulin under stimulatory glucose concentrations. Neither wortmannin (100 nM) nor rapamycin (100 nM) affected glucose-stimulated fractional insulin secretion (data not shown).

Glucose Responsiveness of the beta -Cells-- To determine whether the reduced insulin content and secretion were due to reduced glucose sensitivity, we examined glucose responsiveness of the IRS-1-overproducing beta -cells. Glucose strongly stimulates beta -cell metabolism, and its effect is reflected by the MTT assay (36, 37). As shown in Fig. 3A, addition of 15 mM glucose doubled MTT values in both the beta IRS1-A and the Neo control cells. No significant difference was detected between the two cell lines.


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Fig. 3.   Glucose responsiveness of the beta -cells. A, MTT assay; B, cellular ATP content. Cellular ATP content was measured by HPLC as described under "Materials and Methods" and elsewhere (38). Open bars, 0 mM glucose; filled bars, 15 mM glucose. C, glucose-dependent insulin secretion. Insulin secretion at different glucose concentrations were normalized with the maximal insulin secretion at 30 mM glucose and expressed as a percentage of the maximum. Glucose response curves from the two cell lines were not significantly different (one-way ANOVA test, p > 0.28). All data represent means ± S.E. from at least two independent experiments, each performed in triplicate.

Glucose metabolism also results in the production of ATP. An increased cellular ATP/ADP ratio is a critical event triggering glucose-stimulated insulin secretion. To determine whether IRS-1 overexpression affects the ability of the beta -cell to produce ATP upon glucose stimulation, we measured cellular nucleotide contents using HPLC. As indicated in Fig. 3B, glucose increased cellular ATP content in both Neo control and beta IRS1-A cells: 1.7 ± 0.3- and 2.0 ± 0.4-fold increase respectively. No statistical difference was detected (p > 0.05).

Glucose-stimulated insulin secretion was also measured at different glucose concentrations ranging from 0 to 30 mM. Insulin secretion at 30 mM glucose was set as the maximum. Insulin secretion at other glucose concentrations was expressed as a percentage of the maximum. The data were fitted to generate sigmoidal glucose-response curves for the beta IRS1-A and Neo control cells (Fig. 3C). No significant difference was detected between the two cell lines (two-way ANOVA test, p > 0.28). These data indicate that overexpression of IRS-1 did not change beta -cell glucose sensitivity or its glucose metabolism in general.

Down-regulation of Insulin Biosynthesis at Translational Level-- IRS-1-overproducing cells exhibited a reduced rate of insulin biosynthesis both in the absence or the presence of 16 mM glucose. Addition of 16 mM glucose increased (pro)insulin as well as the total protein biosynthesis in beta -cells (Fig. 4A). [3H]Leucine incorporation into total protein at 0 mM glucose (G0) was 3.4 ± 0.4 × 106 dpm/mg of protein (beta IRS1-A) and 3.0 ± 0.3 × 106 dpm/mg of protein (control), and at 16 mM glucose (G16), it was 5.7 ± 0.5 × 106 dpm/mg protein (beta IRS1-A) and 5.6 ± 0.5 × 106 dpm/mg of protein (control). However, insulin biosynthesis was significantly reduced as judged by insulin-specific fractional biosynthesis (insulin/total trichloroacetic acid-precipitable proteins) (Fig. 4B) at both basal and glucose stimulated states. Insulin biosynthesis was reduced 66% in the beta IRS1-A cells compared with the control. This down-regulation is specific to the translational regulation. Insulin mRNA levels in beta IRS1-A and control cells were similar as measured with the RNA protection assay (42, 43): 100 ± 19% (control) versus 132 ± 60% (beta IRS1-A) (n = 10, p > 0.1). These data demonstrated that overexpression of IRS-1 in beta -cells decreased the rate of insulin biosynthesis at the translational level but did not change the steady-state insulin mRNA level.


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Fig. 4.   Insulin biosynthesis. A, glucose-stimulated insulin biosynthesis. Insulin biosynthesis was measured with [3H]leucine pulse-chase assay. Insulin-specific radioactivity was immunoprecipitated with anti-insulin antibody (15). For each cell line, the rate of insulin-specific [3H]leucine incorporation at 0 mM glucose was set as basal level. With 16 mM glucose stimulation, the rate of insulin biosynthesis increased 2-fold in both cell lines. B, fractional insulin biosynthesis (% of trichloroacetic acid). Insulin-specific 3H-counts were divided by total trichloroacetic acid-precipitable 3H-counts under each condition. Open bars 0 mM glucose; solid bars, 16 mM glucose. Data are presented as mean ± S.E., n = 7. *, p < 0.001.

Increased beta -Cell Cytosol Ca2+ Level-- An increase in intracellular Ca2+ is recognized as a key obligatory step in insulin secretion and has been implicated mechanistically in insulin release (1, 23). Calcium content in ER has been shown to affect insulin biosynthesis. Reduced ER Ca2+ content results in decreased rate of translation initiation (25). It is currently unknown whether insulin receptor signaling affects beta -cell Ca2+ homeostasis. To investigate that, we measured cytosolic Ca2+ levels in beta IRS1-A and control cells using fura-2 Ca2+ indicator as described under "Materials and Methods." As shown in Fig. 5A, cytosolic free [Ca2+] in the beta IRS1-A cells was increased more than 3-fold both in basal (G0) and 15 mM glucose-stimulated (G15) conditions: 278 ± 39 nM (beta IRS1-A) versus 81 ± 18 nM (control) at G0 (p < 0.001) and 739 ± 121 nM(beta IRS1-A) versus 209 ± 42 nM (control) at G15 (p < 0.001). The peak value upon glucose stimulation, however, was not different between the two cell lines: 1886 ± 614 nM (beta IRS1-A) versus 1634 ± 512 nM (control).


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Fig. 5.   Cytosol Ca2+ measurements. Cytosol [Ca2+] was measured as described under "Materials and Methods" and by Gao et al. (37) under the conditions indicated by the perifusion procedure bar shown above the curves. A, effect of IRS-1 on cytosol free [Ca2+]. Cytosol [Ca2+] in beta IRS1-A cells and control cells was measured at 0 mM glucose (G0) for 3 min and then shifted to 15 mM glucose plus 1 mM carbachol (G15+CCH). Data are expressed as mean ± S.E., n = 30. B, effect of the insulin receptor on cytosol free [Ca2+]. The IR-overexpressing cell lines were established as described (15). beta TC6-F7 and NEO are parental and vector control cells, respectively. AK-S2, transfected beta TC6-F7 cells overexpressing the kinase-inactive insulin receptor AK1018; IR-S2, transfectants overexpressing the wild type insulin receptor. For each cell line, at least 30 individual cells were measured in three different experiments. Differences among the cell lines were analyzed by one-way ANOVA. **, p < 0.01. C, the effect of thapsigargin on free cytosol [Ca2+]. The cells were perifused with KRB without glucose (G0) for 1 min, shifted to KRB plus 200 nM thapsigargin (G0+THP) for 5 min, and then incubated with 15 mM glucose, 1 mM carbachol and 200 nM thapsigargin (G15+CCH+THP). Data were obtained from two experiments with at least 20 individual cells measured. D, effect of thapsigargin. Cytosol [Ca2+] for the first 60 s (at the G0 condition in B) and from 240 to 360 s (G0+THP) were averaged and are presented as mean ± S.E.: Cytosol [Ca2+] was significantly different (**, p < 0.01) between two cell lines at 0 mM but not at 200 nM thapsigargin.

To determine whether this increased cytosolic [Ca2+] is specific to insulin receptor signaling, we examined cytosolic Ca2+ levels in the insulin receptor-overproducing cells (Fig. 5B). We have previously established beta -cell lines overproducing the wild type IR (the IR-S2 cells) or the kinase-deficient IR (the AK-S2 cells) (15). Compared with control cells, expression level of IR was 4-fold and 2-fold higher in the IR-S2 and AK-S2 cells, respectively (15). Cytosolic [Ca2+] in control cell lines beta TC6-F7 and NEO was at 90 ± 9 and 88 ± 8 nM, respectively. The cells overexpressing the kinase-deficient insulin receptor (AK-S2) had a [Ca2+] of 92 ± 11 nM similar to the controls. The cells overproducing the wild type IR (IR-S2) had a significantly elevated Ca2+ level: 135 ± 16 nM (p < 0.01 compared with controls, a 50% increase). These data demonstrated that elevated cytosol [Ca2+] is insulin receptor kinase-dependent. Overexpression of IR and IRS-1 both increased cytosol [Ca2+] in beta -cells.

ER Ca2+-ATPase plays an important role in regulating cytosol [Ca2+]. It sequestrates cytosolic Ca2+ into endoplasmic reticulum in an ATP-dependent manner (44-46) therefore lowering [Ca2+]i. Thapsigargin is a specific inhibitor of the ER Ca2+-ATPase (31, 47). Addition of thapsigargin to the beta -cells prevented ER Ca2+ uptake, therefore leading to elevated cytosol free [Ca2+] (32). As shown in Fig. 4C, addition of 200 nM thapsigargin to the control beta -cell beta TC6-F7 led to an increase in [Ca2+]i: from the basal level of 107 ± 7 nM to 184 ± 11 nM (p < 0.05) (Fig. 5, C and D). In the beta IRS1-A cells, however, thapsigargin had no effect on cytosol free [Ca2+]: 223 ± 35 nM (basal) versus 194 ± 36 nM (200 nM thapsigargin). Because addition of thapsigargin raised [Ca2+]i to the same level as that caused by overexpression of IRS-1, these data indicated that altered ER Ca2+ uptake may be the major cause of elevated [Ca2+]i in IRS-1-overproducing cells.

Inhibition of Endoplasmic Reticulum Ca2+ Uptake-- To determine whether endoplasmic reticulum Ca2+ uptake is affected by IRS-1, we directly measured ER Ca2+ uptake with permeabilized beta -cells. Calcium uptake by endoplasmic reticulum can be directly measured with radioactive 45Ca2+. We assayed ER Ca2+ uptake at two Ca2+ concentrations: 100 nM (basal condition) and 500 nM (equivalent to glucose-stimulated beta -cells). As shown in Fig. 6, Ca2+ uptake in beta IRS1-A was significantly reduced at both Ca2+ concentrations compared with control. At 100 nM [Ca2+], ER Ca2+ uptake for the control and beta IRS1-A cell was 4.28 ± 0.89 and 2.77 ± 0.22 nmol/mg of protein, respectively (a reduction of 35%) (p < 0.05). At 500 nM [Ca2+], the uptake was 11.42 ± 1.65 (control) and 7.15 ± 0.90 (beta IRS1-A) nmol/mg of protein (p = 0.01), a reduction of 37%. Similar ER Ca2+ uptake results were also obtained from two additional IRS-1-overproducing clones (data not shown). These data clearly demonstrated that overexpression of IRS-1 reduced endoplasmic reticulum Ca2+ uptake and therefore lowered ER Ca2+ content.


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Fig. 6.   Endoplasmic reticulum Ca2+ uptake. ATP-dependent endoplasmic reticulum Ca2+ uptake was measured with permeabilized beta -cells as described under "Materials and Methods." Open bars, the NEO control cells; solid bars, beta IRS1-A cells. Data shown are mean ± S.E. from two experiments each in triplicate. *, p = 0.04; **, p = 0.01.

Inhibition of beta -Cell Proliferation-- The insulin receptor signaling pathway is also implicated in mitogenic regulation (49, 50). To determine how overexpression of IRS-1 affects beta -cells growth, we used the [3H]thymidine incorporation assay (51) to assess beta -cell proliferation (Fig. 7). beta IRS1-A cells exhibited a 32 ± 4% (n = 6) decrease in the rate of cell proliferation as measured by [3H]thymidine incorporation (6,842 ± 513 dpm/µg DNA) compared with the passage-matched beta TC6-F7 control cells (10,114 ± 890 dpm/µg DNA) (p = 0.02) in the presence of 10% serum. In the absence of serum, the growth rate of beta IRS1-A was reduced 40% compared with the control (1,895 ± 167 dpm/µg DNA, beta IRS1-A versus 3,183 ± 274 dpm/µg DNA, control; n = 6) (p = 0.007). No increase in beta -cell apoptosis was observed when measured with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (52) (data not shown). These data demonstrated that IRS-1 negatively regulates beta -cell proliferation.


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Fig. 7.   beta -Cell proliferation assay. beta -Cell proliferation was determined by incorporation of [3H]thymidine in the presence (solid bars) or the absence (open bars) of 10% fetal bovine serum (FBS). Data are shown as mean ± S.E., n = 12. *, p = 0.02; **, p = 0.007.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Earlier studies had suggested that the insulin receptor may be present in beta -cells and that it could function to regulate insulin secretion. For many years, however, this concept was viewed as controversial in the absence of definitive evidence identifying the insulin receptor in the beta -cells. Our group has recently demonstrated that the various components of the insulin receptor signaling pathway are indeed present in beta -cells, including the insulin receptor and IRS-1 (12, 13). Furthermore, we had shown that glucose-induced insulin secretion activates the beta -cell surface insulin receptor tyrosine kinase and its intracellular signal transduction pathway and had proposed that this represented a novel autocrine mechanism for the regulation of beta -cell function (12). However, the physiological role of this pathway in the beta -cell has been difficult to elucidate because the beta -cell is not a classical insulin target tissue, and there is scant evidence that insulin regulates its own secretion. Very recently, it has been shown that one role of the insulin receptor signaling pathway in beta -cells is regulation of beta -cell growth because disruption of IRS-2 leads to beta -cell deficiency at birth and diabetes, and it has been proposed that IRS-2-dependent signaling pathways are involved in beta -cell neogenesis, proliferation, and survival (17). In contrast, mice heterozygous for null alleles of the insulin receptor and IRS-1 become diabetic and develop beta -cell hyperplasia (21). Other studies have also shown that insulin receptor signaling in the beta -cell can regulate insulin gene transcription, as well as autoregulation of protein synthesis via PHAS-1 phosphorylation (14, 16). Thus, the insulin receptor signaling pathway of the beta -cell appears to have multiple physiological effects.

To identify the role of IRS-1 in insulin secreting beta -cells, we overexpressed IRS-1 in a clonal beta -cell line beta TC6-F7. Our data demonstrates that IRS-1 is involved in regulating Ca2+ homeostasis, insulin secretion, insulin biosynthesis, and beta -cell proliferation and that elevated expression of IRS-1 induces beta -cell failure. This is the first study to demonstrate that a 2-fold overexpression of IRS-1 in beta -cells increases beta -cell cytosol Ca2+ levels and reduces ER Ca2+ content. These findings are significant because the ER is one of the major compartments for intracellular Ca2+ storage. Elegant earlier experiments have shown that the ER is actively involved in regulating intracellular Ca2+ in the nanomolar range (27, 28). In beta -cells, it is widely established that glucose stimulation results in an increase in intracellular Ca2+, a required step for insulin secretion. Typically, basal Ca2+ concentrations are in the 80-100 nM range, and following glucose stimulation, they increase 3-5-fold. Once the stimulation is removed, the ER sequesters excess cytosolic Ca2+, and the Ca2+ levels return to baseline. The ER Ca2+-ATPase is the main pump responsible for Ca2+ uptake into the ER. Its biochemical characteristics have been extensively described, and it is implicated in the regulation of intracellular Ca2+ homeostasis. Recently, two isoforms of the Ca2+-ATPase, SERCA2 and SERCA3, have been localized to the islet. Furthermore, SERCA3 expression is reduced in the GK rat, a nonobese model of type 2 diabetes (44-46).

Our observations that thapsigargin, an ER Ca2+-ATPase-specific inhibitor, increased cytosolic Ca2+ levels in the control cells, but not in the IRS-1-overproducing cells (Fig. 5, C and D) suggest that ER Ca2+-ATPase in the beta IRS1-A cells could have been suppressed by IRS-1 overproduction. This is also strongly supported by the observed decrease in beta IRS1-A ER Ca2+ uptake (an indirect measurement of Ca2+-ATPase) and the fact that Ca2+ release from the ER (induced by A23187, data not shown) is not changed in beta IRS1-A cells. A possible explanation for IRS-1-induced inhibition of ER Ca2+-ATPase is based on an elegant study by Kahn and co-workers (33), who have identified SERCA1 in skeletal muscle and SERCA2 in cardiac muscle as novel IRS-1- and IRS-2-binding proteins. Importantly, this interaction is insulin-dependent (maximal at 100 nM insulin and at 5 min of stimulation), and requires tyrosine phosphorylation of IRS-1. Whether there is a similar interaction between IRS-1 and the beta -cell ER Ca2+-ATPase still remains to be determined, although our data suggest that such an interaction could result in decreased ER Ca2+-ATPase activity, leading to reduced ER Ca2+ uptake in beta -cells and increased cytosolic Ca2+ concentrations.

Our study provides a novel functional link between the IRS-1 signaling pathway and the stimulus-secretion pathway in beta -cells, which we believe to be physiologically significant. We postulate that under basal conditions in the beta -cell this pathway is not activated. However, once glucose or other secretagogues stimulate insulin secretion, the released insulin will feed back to the beta -cell insulin receptor and activates the associated signal transduction pathway. Increased signaling results in IRS-1 tyrosine phosphorylation (12) and subsequent inhibition of ER Ca2+ uptake as shown by this study. Decreased Ca2+ fluxes into the ER can then increase cytosolic Ca2+ and further facilitate the maintenance of increased Ca2+ levels due to secretagogue-induced Ca2+ influx from the extracellular space. We believe that our studies have identified a novel pathway of autocrine regulation of intracellular Ca2+ homeostasis and insulin secretion in the beta -cell of the endocrine pancreas.

Insulin secretion and cellular insulin content were significantly reduced in the cells overproducing IRS-1. This is most likely the consequence of reduced insulin biosynthesis. Elevated expression of IRS-1 inhibits insulin biosynthesis at the translational level as measured by the [3H]leucine labeling assay. This inhibition is likely due to the reduced ER [Ca2+] found in the IRS-1-overproducing cells. A similar finding that ER Ca2+ affects insulin biosynthesis has been reported before (25, 26). The insulin mRNA level in the IRS-1-overexpressing cells was similar to the controls as measured with RNA protection assay. It suggests that IRS-1 may not be involved in mediating activation of insulin gene transcription in beta IRS1-A cells. A recent report suggests that IRS-2 may mediate signals for insulin gene transcription (16).

Augmented expression of IRS-1 also leads to inhibition of beta -cell growth. beta IRS1-A cells exhibited a 32 ± 4% decrease in the rate of cell proliferation as measured by [3H]thymidine incorporation compared with the control. These observations are supported by the finding that loss of IRS-1 function leads to beta -cell hyperplasia and hyperinsulinemia in the IRS-1 knockout animals (21, 48). beta -Cell function is also regulated by the insulin receptor substrate IRS-2. Loss of IRS-2 function results in beta -cell failure, including reduced beta -cell growth and decreased insulin secretion. Deletion of IRS-2 leads to development of diabetes in transgenic mice (4, 17). It appears that insulin receptor signaling differentially regulates beta -cell function via different substrates. The net output of insulin receptor signaling in beta -cells may therefore depend on the relative strength of different substrate signals. Abnormalities in the insulin receptor substrates, e.g. altered IRSs expression levels, may directly contribute to beta -cell failure in diabetes.

    ACKNOWLEDGEMENT

We thank Konrad Talbot for critical reading of the manuscript.

    FOOTNOTES

* Portions of this work were presented at the American Diabetes Association 58th Annual Scientific Sessions in June 1998 (Chicago, IL). This study was supported by National Institutes of Health Grants DK43354 and DK49814. The Diabetes Endocrinology Research Center Radioimmunoassay Core is supported by National Institutes of Health 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 Recipient of NIH Research Career Development Award K04 DK02217. To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 230 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104. Tel.: 215-898-0025; Fax: 215-573-2266; E-mail: wolfb{at}mail.med.upenn.edu.

    ABBREVIATIONS

The abbreviations used are: IR, insulin receptor; IRS, IR substrate; ER, endoplasmic reticulum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ANOVA, analysis of variance; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; KRB, Krebs-Ringer buffer; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase.

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