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Insulin Potentiates Ca²⁺ Signaling and Phosphatidylinositol 4,5-Bisphosphate Hydrolysis Induced by G_q Protein-Coupled Receptor Agonists through an mTOR-Dependent Pathway

Unit of Signal Transduction and Gastrointestinal Cancer, Division of Digestive Diseases, Department of Medicine, University of California at Los Angeles-Center for Ulcer Research and Education, Digestive Diseases Research Center and Molecular Biology Institute, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095

Address all correspondence and requests for reprints to: Enrique Rozengurt, Department of Medicine, David Geffen School of Medicine, 900 Veteran Avenue, Warren Hall Room 11-124, University of California at Los Angeles, Los Angeles, California 90095-1786. E-mail: erozengurt{at}mednet.ucla.edu.

	Abstract

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Multiple lines of evidence support the existence of crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling systems. However, the precise molecular mechanism(s) mediating this interaction is poorly understood. The results presented in this study show that exposure of ductal pancreatic adenocarcinoma BxPc-3, HPAF-II, and PANC-1 cells to insulin for as little as 1 min rapidly enhanced the magnitude and the rate of increase in intracellular Ca²⁺ concentration produced by the GPCR agonists bradykinin, angiotensin II, vasopressin, neurotensin, and bombesin. The potentiating effect of insulin was dose dependent, and it was produced in response to G_q protein-coupled, but not G_i protein-coupled, receptor agonists. Real-time imaging of single cells showed that treatment with insulin enhances the rate and magnitude of phosphatidylinositol 4,5-bisphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in response to GPCR stimulation. Short-term treatment with rapamycin, an mTOR (mammalian target of rapamycin) inhibitor, completely abrogated the ability of insulin to increase the rate and magnitude of Ca²⁺ signaling and production of inositol 1,4,5-trisphosphate in response to bradykinin stimulation, indicating that insulin potentiates G_q protein-coupled receptor signaling through an mTOR-dependent pathway. We propose that the potentiation of GPCR signaling by insulin provides a mechanism by which insulin enhances cellular responsiveness to G_q protein-coupled receptor agonists, including GPCR-mediated autocrine and paracrine loops in cancer cells.

	Introduction

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MULTIPLE LINES OF experimental, epidemiological, and clinical evidence support the notion that a crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling systems plays a critical role in the regulation of normal physiological functions as well as in the pathogenesis of a variety of abnormal processes (1). For example, genetic disruption of the bradykinin B₂ receptor in diabetic Akita mice markedly increases the severity of the diabetic phenotype, implying that a crosstalk between insulin and bradykinin plays a critical role in normal homeostatic regulation (2, 3). An important role of a crosstalk between insulin and angiotensin II (ANG II) in the pathogenesis of cardiovascular and renal pathologies in obesity, metabolic syndrome, and type II diabetes is increasingly recognized (2, 3, 4, 5, 6) and underscored by the clinical benefits of angiotensin-converting enzyme (ACE) inhibition in these conditions (7, 8). Epidemiological studies have also linked hyperinsulinemia and type II diabetes with increased risk for developing a variety of clinically aggressive cancers of several tissues (9, 10, 11, 12), including pancreatic ductal adenocarcinoma (13, 14, 15), colon (16, 17, 18), and prostate (19), and administration of ACE inhibitors protect against various cancers (20, 21). Interestingly, many cancer cells, including human pancreatic adenocarcinoma cells, are known to express multiple functional GPCRs that mediate rapid signaling and mitogenesis in response to their corresponding agonists (22, 23, 24, 25, 26, 27, 28), and, accordingly, GPCRs are increasingly implicated in autocrine/paracrine growth stimulation of cancer cells (29, 30, 31, 32, 33, 34, 35). It is plausible, therefore, that a crosstalk between insulin and GPCR signaling pathways could also exist in cancer cells.

The signaling pathways activated by either GPCR agonists or insulin have been the subject of intense scrutiny (8, 30, 31, 36, 37, 38). Many agonists, including ANG II and bradykinin, bind to their cognate heptahelical GPCRs and activate pertussis toxin (PTx)-insensitive G_q, promoting its dissociation into G_q and Gß and the exchange of GDP bound to G_q for GTP. The resulting GTP-G_q complex activates the ß isoforms of phospholipase C (PLC) via interaction with the carboxy-terminal region of the enzyme (39). PLC catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] to produce two-second messengers: inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and diacylglycerol (DAG). Ins(1,4,5)P₃ binds to its intracellular receptor, a ligand-gated Ca²⁺ channel located in the endoplasmic reticulum (40), and triggers the release of Ca²⁺ from internal stores, leading to a rapid and transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), whereas DAG stimulates members of the protein kinase C (PKC) and D (PKD) families (30, 41, 42, 43). Binding of insulin to its tetrameric receptor results in receptor autophosphorylation and activation of the receptor tyrosine kinase, followed by tyrosine phosphorylation of insulin receptor substrates that further propagate downstream signals (36, 44). The phosphatidylinositol 3 (PI3)-kinase/Akt/mTOR (mammalian target of rapamycin) signaling module is one of the major insulin-induced pathways. Recent evidence indicates that mTOR is a point of convergence of signals from mitogenic growth factors, nutrients, cellular energy levels, and stress conditions to stimulate protein synthesis and cell growth (45). Despite the clinical and biological importance of the crosstalk between insulin receptor and GPCR signaling systems, especially those activated by the peptides regulated by ACE (ANG II and bradykinin), little is known about mechanisms by which insulin receptor activation rapidly modulates signaling through G_q protein-coupled receptors (G_qPCRs).

Using pancreatic adenocarcinoma cells that express multiple GPCRs as a model system, we report here that cell exposure to insulin rapidly and selectively increases the rate and magnitude of the increase in [Ca²⁺]_i elicited by multiple G_qPCR agonists, including ANG II, bradykinin, bombesin, neurotensin, and vasopressin. Using real-time imaging of changes in PtdIns(4,5)P₂ hydrolysis and generation of Ins(1,4,5)P₃ in single cells, we found that treatment with insulin enhances the rate and magnitude of these second-messenger-generating responses. Our results demonstrate that insulin-induced potentiation of G_qPCR signaling is mediated through a rapamycin-sensitive, PI3-kinase/mTOR-dependent pathway.

	Materials and Methods

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Cell culture
In view of the phenotypic heterogeneity of pancreatic cancer cell lines manifested in different growth and differentiation patterns, oncogene, and GPCR expression (22), the experiments presented here were performed in different pancreatic cell lines originated in different laboratories. The following human pancreatic cancer cell lines, obtained from American Type Culture Collection (Manassas, VA), were used: BxPc-3, HPAF-II, and PANC-1. BxPc-3 and PANC-1 are less well-differentiated cell lines, whereas HPAF-II is a well-differentiated cell line derived from a human pancreatic adenocarcinoma. Cells were grown in 10-cm tissue culture dishes in a 37 C incubator with a humidified atmosphere as follows: BxPc-3 and HPAF-II in 5% CO₂, PANC-1 in 10% CO₂. BxPc-3 and HPAF-II cells were grown in MEM (Invitrogen, Carlsbad, CA) supplemented with 0.1 mM MEM nonessential amino acids, antibiotics (100 U/ml penicillin plus 50 mg/ml streptomycin and gentamycin), and 10% fetal bovine serum (FBS). PANC-1 were grown in DMEM (Invitrogen) with 4 mM glutamine, 1 mM Na-pyruvate, antibiotics (100 U/ml penicillin plus 50 mg/ml streptomycin and gentamycin), and 10% FBS. To determine whether the results obtained in pancreatic cancer cells can replicated in cultures of nontumorigenic epithelial cells, we used intestinal crypt-derived IEC-18 cells, a cell line used in studies of GPCR-induced signal transduction (46, 47, 48). These cells were grown in DMEM (Invitrogen) with 4 mM glutamine, 1 mM Na-pyruvate, antibiotics (100 U/ml penicillin plus 50 mg/ml streptomycin and gentamycin), and 5% FBS.

Measurement of [Ca²⁺]_i
Cells grown on glass coverslips for 4–5 d were washed in Hanks’ balanced salt solution supplemented with 0.03% NaHCO₃, 1.3 mM CaCl₂, 0.5 mM MgCl₂, 0.4 mM MgSO₄, and 0.1% BSA (pH 7.4) (Hanks’ buffer). After washing, cells were incubated with 5 µM fura 2-tetra-acetoxy methyl ester (fura 2-AME) from a stock of 1 mM in dimethylsulfoxide for 10 min in a 37 C incubator. Cells were then washed again with Hanks’ buffer and left at room temperature for an additional 5 min. The fura-loaded cells were introduced in a cuvette containing the incubation medium (Hanks’ buffer), and the cuvette was placed into a Hitachi (Tokyo, Japan) F-2000 fluorospectrophotometer. The incubation medium in the cuvette was continuously stirred at 37 C. The excitation wavelengths were set at 340 and 380 nm, and the emission wavelength was set at 510 nm. The maximum fluorescence was determined after membrane permeabilization by the addition of 37.5 µM digitonin. The minimum fluorescence was measured after the Ca²⁺ in the solution was chelated by the addition of EGTA at a final concentration of 25 mM. A K_d of 224 nM was used for the Ca²⁺ dissociation constant from fura 2 in the cells at 37 C. [Ca²⁺]_i was determined automatically by the cation measurement software of the F-2000 fluorospectrophotometer. Agonists and/or antagonists were added at various time points during recording, as indicated in the individual experiments.

Cell transfection
BxPc-3 cells were transfected with the plasmid containing a cDNA encoding a green fluorescent protein (GFP) tagged-pleckstrin homology domain (PHD) of human PLC-1 (GFP-PHD) (49) by using Lipofectin or Lipofectamine Plus (Invitrogen) as suggested by the manufacturer. Analysis of the cells transiently transfected were performed 24 h after transfection.

Real-time Ins(1,4,5)P₃ imaging in single live cells
Single live-cell imaging of the fluorescent biosensor for Ins(1,4,5)P₃ (GFP-PHD) was achieved with a fluorescence microscope. The plasmid encoding GFP-PHD was produced as described previously (49). To maintain a constant temperature of 37 C during the experimental procedures, cells grown in the 15-mm glass coverslips were mounted in a RC-25 perfusion chamber (Warner Instruments, Hamden, CT) and perfused with medium preheated at 37 C by a TC-344B chamber system heater controller (Warner Instruments). The medium was supplemented with 10 mM HEPES (pH 7.2). The microscope used was a Zeiss (Oberkochen, Germany) epifluorescent Axioskop with a Zeiss Achroplan 40x/0.75 water immersion objective (Zeiss). Images were captured as uncompressed 24-bit TIFF files with a SPOT cooled single CCD color digital camera driven by SPOT version 2.1 software (Diagnostic Instruments, Sterling Heights, MI). GFP fluorescence was observed with a HI Q filter set for fluorescein isothiocyanate (Chroma Technology, Rockingham, VT).

Live-cell imaging and quantitative analysis of the relative change in plasma membrane and cytosol fluorescence intensity of individual cells were performed using Sigmascan version 3.0 (SPSS, Chicago, IL) as described in our previous paper (49). We analyzed 60 cells in each experiment, and each experiment was performed in at least triplicate. The selected cells displayed in the figures were representative of 90% of the population of positive cells.

Western blot analysis
Samples of cell lysates were directly solubilized by boiling in SDS-PAGE sample buffer [200 mM Tris-HCl (pH 6.8), 2 mM EDTA, 0.1 M Na₃VO₄, 6% SDS, 10% glycerol, and 4% 2-mercaptoethanol]. Proteins were resolved in SDS-10% PAGE and transferred to Immobilon-P membranes (Millipore, Billerica, MA), as described previously (26), and blocked for 2 h with 5% nonfat dried milk in PBS (pH 7.2). Membranes were incubated overnight with a polyclonal antibody that detects the phosphorylated state of myristoylated alanine-rich C kinase substrate (MARCKS) at serine152/156 (Cell Signaling Technology, Danvers, MA) or anti-phospho-PKD-2 (serine 876) polyclonal antibody (Millipore) at a dilution of 1:1000 in PBS containing 3% nonfat dried milk and 0.5% Tween 20. Immunoreactive bands were visualized using horseradish peroxidase-conjugated antirabbit IgG and enhanced chemiluminescence (GE Healthcare Biosciences, Little Chalfont, Buckinghamshire, UK) detection. The same membranes were subsequently stripped and probed in a similar manner with anti-MARCKS (M-20) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:500 and anti-PKCµ/PKD (C-20), which recognizes PKD and PKD2 (Santa Cruz Biotechnology) at a dilution of 1:1000 in PBS containing 5% nonfat dried milk and 0.1% Tween 20. Autoradiograms were scanned, and the labeled bands were quantified using the Quantity One software program (Bio-Rad, Hercules, CA).

Statistical analysis
The values obtained are presented as the mean ± SEM and analyzed with Student’s t test, using SigmaPlot 2000 (SPSS).

Materials
Bradykinin, ANG II, bombesin, neurotensin, and vasopressin were obtained from Sigma (St. Louis, MO). U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene], LY-294002 [2-(4-morpholinyl)-8-phenyl-(4H)-1-benzopyran-4-one], wortmannin, and rapamycin were from Calbiochem (La Jolla, CA). All other reagents were of the highest grade commercially available.

	Results

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Insulin potentiates Ca²⁺ signaling by G_qPCR agonists in human pancreatic adenocarcinoma cells
The mobilization of Ca²⁺ from intracellular stores leading to a rapid and transient increase in [Ca²⁺]_i is one of the earliest events stimulated by agonist binding to GPCRs that signal through G_q-mediated activation of PLC. To determine whether insulin modulates GPCR-mediated increase in [Ca²⁺]_i, human pancreatic cancer BxPc-3 cells loaded with the fluorescent Ca²⁺ indicator fura 2-AME were stimulated with the GPCR agonist bradykinin, and the changes in [Ca²⁺]_i were continuously recorded as described in Materials and Methods.

Addition of bradykinin (5 nM) to BxPc-3 cells induced a rapid increase in [Ca²⁺]_i from a basal level of 185 ± 6 nM (mean ± SEM; n = 122) to a peak value of 601 ± 12 nM (n = 120) at 25–35 sec, which subsequently declined toward a plateau phase (Fig. 1A). Addition of insulin (10 ng/ml) to BxPc-3 cells did not produce any detectable change in [Ca²⁺]_i (Fig. 1A). However, exposure of BxPc-3 cells to 10 ng/ml insulin for 5 min before their stimulation with bradykinin promoted a striking enhancement in [Ca²⁺]_i that reached a maximum peak of 1122 ± 84 nM (n = 109) (Fig. 1A). The rate of [Ca²⁺]_i rise after the addition of bradykinin was markedly increased, implying that exposure to insulin also accelerates GPCR-elicited intracellular Ca²⁺ mobilization (Fig. 1A).

View larger version (17K): [in this window] [in a new window]   FIG. 1. Exposure to insulin enhances the peak and rate of the increase in [Ca2+]i induced by bradykinin, ANG II, or vasopressin in BxPc-3 cells. BxPc-3 cells loaded with the Ca2+ indicator fura 2-AME were treated with (thick traces) or without (thin traces) 10 ng/ml insulin for 5 min and then stimulated with 5 nM bradykinin (BK) (A, left), 100 nM ANG II (B, left), or 100 nM vasopressin (VP) (C, left). [Ca2+]i was monitored as described in Materials and Methods. Insulin did not induce an increase in [Ca2+]i or affect the basal levels of [Ca2+]i as shown in A (insulin, thick trace). Right columns, Peak, Maximal increment in [Ca2+]i in response to bradykinin (BK) (A), ANG II (B), or vasopressin (VP) (C) in cells incubated without (open bars) or with (filled bars) 10 ng/ml insulin for 5 min before the GPCR agonist stimulation. Rate, The slope of the trace of increase in [Ca2+]i after the addition of bradykinin (BK) (A), ANG II (B), and vasopressin (VP) (C) in the absence (open bars) or presence (filled bars) of insulin pretreatment. The units represent the increment in [Ca2+]i during the first 10 sec from the moment that [Ca2+]i started to increase. The panels with bradykinin represent 20 independent experiments (summarized n = 122 for bradykinin; n = 109 for bradykinin after insulin pretreatment), and the panels with ANG II and vasopressin represent at least five independent experiments for each agonist (n = 35 for ANG II, n = 40 for ANG II after insulin pretreatment; n = 32 for vasopressin and n = 36 for vasopressin after insulin pretreatment). The results shown are the mean ± SEM and compared with the control (absence of insulin pretreatment). **, P < 0.01.

Insulin pretreatment enhanced the increase in [Ca²⁺]_i in response to either bradykinin or ANG II in a concentration-dependent manner (Fig. 2A). The potentiating effect of insulin could be detected at a concentration as low as 1 ng/ml (0.17 nM), with a half-maximal stimulatory concentration achieved at approximately 5 ng/ml (0.85 nM). Interestingly, these concentrations are in good agreement with the range of circulating insulin levels observed in fasting normal insulin-sensitive people (0.1 nM) and in subjects with insulin resistance and in type 2 diabetes (1.5 nM) (50). We also determined the length of time required by insulin to enhance the increase in [Ca²⁺]_i in response to G_qPCR stimulation. Maximal potentiation of [Ca²⁺]_i by bradykinin or ANGII was achieved after 1–2 min of previous exposure to insulin (Fig. 2B). Addition of 10 ng/ml insulin after cell stimulation with bradykinin did not produce any additional increase in [Ca²⁺]_i (results not shown).

View larger version (12K): [in this window] [in a new window]   FIG. 2. Insulin enhances [Ca2+]i increase induced by bradykinin in a dose- and time-dependent manner in BxPc-3 cells and potentiates Ca2+ signaling in response to multiple GPCR agonists in other human pancreatic cancer cell lines. BxPc-3 cells were pretreated with increasing concentrations of insulin for 5 min (A) or for increasing lengths of time (2.5 sec to 5 min) with 10 ng/ml insulin (B). The cells then were stimulated with either 5 nM bradykinin (BK) or 100 nM ANG II. In all cases, [Ca2+]i was measured as described in Materials and Methods. Each point is representative of at least three independent experiments performed in triplicate. In A, the x-axis at the bottom shows the concentration of insulin in nanograms per milliliter, whereas at the top, it shows the concentrations in nanomolar. C, HPAF-II cells were treated without (open bars) or with (filled bars) 10 ng/ml insulin for 5 min and then challenged with 10 nM neurotensin (NT), 10 nM ANG II, 10 nM bombesin (BM), or 10 nM bradykinin (BK), and the [Ca2+]i was measured. The panels represent at least four independent experiments (n = 24 for neurotensin, n = 22 for neurotensin after insulin pretreatment; n = 22 for ANG II, n = 26 for ANG II after insulin pretreatment; n = 12 for bombesin, n = 12 for bombesin after insulin pretreatment). D, PANC-1 cells were treated without (open bars) or with (filled bars) 10 ng/ml insulin for 5 min and then challenged with 5 nM neurotensin. [Ca2+]i was measured as described in Materials and Methods. The panel represents four independent experiments (n = 25 for neurotensin; n = 25 for neurotensin after insulin pretreatment.) The values obtained in different conditions are presented as the mean ± SEM and are compared with the control (absence of insulin pretreatment). **, P < 0.01.

Insulin selectively potentiates Ca²⁺ signaling in response to G_qPCR agonists
Because the results illustrated in Figs. 1 and 2 used GPCR agonists that bind to receptors coupled to G_q, it was reasonable to hypothesize that insulin potentiates signaling through G_q/PLCß. As a first step to examine this hypothesis, we determined whether insulin potentiates Ca²⁺ mobilization from internal stores in response to G_qPCR agonists but fails to enhance Ca²⁺ signaling produced by agents that either bypass receptor-mediated pathways or agonists that activate PLC through G_i rather than G_q.

Bradykinin increased [Ca²⁺]_i in BxPc-3 cells by two mechanisms: the initial peak resulted from Ca²⁺ mobilization from intracellular stores because it still occurred after chelation of extracellular Ca²⁺ with EGTA, whereas the plateau phase was mediated by the influx of extracellular Ca²⁺ because it was greatly reduced by chelating extracellular Ca²⁺ with EGTA. As shown in Fig. 3A, the increase in Ca²⁺ signaling produced by insulin pretreatment was maintained after chelation of extracellular Ca²⁺ with EGTA, indicating that insulin enhances bradykinin-induced mobilization of Ca²⁺ from internal stores.

View larger version (12K): [in this window] [in a new window]   FIG. 3. Insulin potentiates bradykinin-induced Ca2+ mobilization from internal stores but does not enhance Ca2+ signaling in response to either thapsigargin or LPA. A, Exposure to insulin enhances Ca2+ mobilization in response to bradykinin. BxPc-3 cells were treated without (left) or with (right) 10 ng/ml insulin for 5 min (insulin pretreatment) and then challenged with 5 nM bradykinin (BK). EGTA (1.5 mM final concentration in the cuvette) was added 10 sec before bradykinin. The free Ca2+ concentration calculated with Maxchelator program (http://www.stanford.edu/cpatton/maxc.html) (53 ) was 421 nM in the cuvette after the addition of EGTA. The tracings are representative of two independent experiments (n = 7). B, Effect of PTx on LPA-induced [Ca2+]i increase. BxPc-3 cells were pretreated with 100 ng/ml PTx (thick traces) or vehicle (thin traces) for 3 h and then stimulated with 500 nM LPA. The tracings are representative of three independent experiments, each performed at least in triplicate. C, Exposure to insulin does not enhance Ca2+ signaling in response to LPA. BxPc-3 cells were treated without (open bars) or with (filled bars) 10 ng/ml insulin for 5 min and then challenged with 500 nM LPA. The results shown are the mean ± SEM of at least three independent experiments (n = 15 for LPA; n = 21 for LPA after insulin pretreatment). D, Exposure to increasing concentrations of insulin does not enhance Ca2+ signaling in response to LPA. BxPc-3 cells were treated with increasing concentrations of insulin for 5 min and then stimulated with 500 nM LPA. E, Exposure to insulin for various times does not enhance Ca2+ signaling in response to LPA. BxPc-3 cells were treated with 10 ng/ml insulin for increasing lengths of time (2.5 sec to 5 min). Then, the cells were stimulated with 500 nM LPA. Each point is representative of at least three independent experiments performed in triplicate.

Lysophosphatidic acid (LPA) binds to heptahelical receptors that induce Ca²⁺ signaling via G_q and/or G_i in different cell types. Addition of 500 nM LPA to BxPc-3 cells stimulated a rapid and transient increase in [Ca²⁺]_i (Fig. 3B) with a peak value of 398 ± 11 nM (n = 25). Treatment of BxPc-3 cells with PTx, which catalyzes ADP ribosylation and inactivation of G proteins of the G_i/G_o family, completely blocked the increase in [Ca²⁺]_i induced by subsequent LPA stimulation in these cells (Fig. 3B). In contrast, PTx did not interfere with Ca²⁺ signaling induced by bradykinin stimulation of BxPc-3 cells (results not shown). Thus, in BxPc-3 cells, LPA induces an increase in [Ca²⁺]_i through G_i, whereas bradykinin elicits Ca²⁺ signaling via a PTx-insensitive GPCR.

In contrast to the results obtained when BxPc-3 cells were challenged with G_qPCR agonists, pretreatment with 10 ng/ml insulin for 5 min failed to potentiate the [Ca²⁺]_i response induced by LPA (Fig. 3C). Furthermore, pretreatment of BxPc-3 cells with different concentrations of insulin (1–1000 ng/ml) for 5 min (Fig. 3D) or with a fixed concentration of insulin (10 ng/ml) for different times (Fig. 3E) failed to enhance LPA-induced Ca²⁺ signaling, even at the highest insulin concentration tested. These results indicate that insulin does not potentiate Ca²⁺ mobilization elicited by agonists that act through this G_i-stimulated pathway.

Insulin potentiates bradykinin-induced Ins(1,4,5)P₃ synthesis in BxPc-3 cells
Ins (1, 4, 5)P₃ is one of the best characterized second messengers that triggers the release of Ca²⁺ from internal stores in response to agonists that stimulate G_q/PLCß. Accordingly, we determined whether insulin receptor signaling potentiates the ability of G_qPCRs to stimulate the synthesis of Ins(1,4,5)P₃ by examining the dynamic distribution of a fusion protein between GFP and the PHD of human PLC-1 (GFP-PHD). This PHD binds PtdIns(4,5)P₂ in the plasma membrane but translocates to the cytosol in response to PLC-mediated Ins(1,4,5)P₃ synthesis (49, 53, 54).

Before GPCR stimulation, GFP-PHD was predominantly localized at the plasma membrane (n = 122) of BxPc-3 cells (Fig. 4A, arrows), whereas GFP was localized throughout the cell (data not shown). Bradykinin stimulation induced a time-dependent translocation of GFP-PHD from the plasma membrane to the cytoplasm (n = 62) (Fig. 4B), reflecting PtdIns(4,5)P₂ hydrolysis and production of Ins(1,4,5)P₃. Although pretreatment with 10 ng/ml insulin for 5 min failed to promote plasma membrane dissociation of GFP-PDH (Fig. 4C, 0 sec), bradykinin stimulation of insulin-pretreated cells caused a very rapid plasma membrane dissociation and cytoplasmic translocation of GFP-PDH (Fig. 4C). Quantitative analysis of the dynamic distribution of GFP-PDH shows that exposure of the cells to insulin before bradykinin stimulation promotes within 10 sec the translocation of GFP-PHD from the plasma membrane to the cytosol (Fig. 4D). Furthermore, previous exposure to insulin significantly increased the extent of plasma membrane dissociation of the Ins(1,4,5)P₃ sensor (Fig. 4, compare B and D). These results indicate that cell exposure to insulin before bradykinin increased the rate and extent of PLC-mediated PtdIns(4,5)P₂ hydrolysis and production of Ins(1,4,5)P₃.

View larger version (17K): [in this window] [in a new window]   FIG. 4. Exposure to insulin potentiates Ins(1,4,5)P3 synthesis in response to bradykinin stimulation. BxPc-3 cells were transiently transfected with a plasmid encoding a fusion protein between GFP and the PH domain of PLC-1 (GFP-PHD). The cultures were perfused with RPMI (containing 5 mM HEPES) and changed to RPMI containing 5 nM bradykinin (and HEPES) during the indicated times. In the insulin-pretreated group, cells were perfused with RPMI (with HEPES) containing 10 ng/ml insulin for 5 min before switching to the RPMI (with HEPES) perfusion fluid containing 10 ng/ml insulin and 5 nM bradykinin. The intracellular distribution of GFP-PHD was monitored under a fluorescence microscope (magnification, x400) (A and C). Culture conditions for live-cell imaging and quantitative analysis (B and D) of the relative change in plasma membrane (M) and cytosol (C) fluorescence intensity of individual cells was performed as described in Materials and Methods. The values obtained at different times in the BK with (D) or without (B) insulin pretreatment were compared with either their correspondent control (time 0) or to each other at each time point. *, P < 0.05; **, P < 0.01, statistical significances in the comparison of the effect of the treatments with their corresponding control (time 0). #, P < 0.05, the statistical significances of the effect of insulin pretreatment vs. no pretreatment at each time point. All the results were obtained by analyzing 60 cells per experiment, with each experiment performed at least in triplicate. The selected cells displayed in the figures were representative of 90% of the population of positive cells.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Insulin enhances MARCKS phosphorylation and PKD2 activation induced by bradykinin in BxPc-3 cells. Confluent BxPc-3 cells were washed twice with Dulbecco’s PBS and incubated in serum-free RPMI for 12 h. Serum-starved cells were pretreated without or with 10 ng/ml insulin for 5 min (indicated as insulin pretreatment) and then stimulated with 5 nM bradykinin (BK) for the indicated time periods at 37 C. The cells were then washed in cold PBS, lysed in 2x SDS-PAGE sample buffer, were run in an SDS-10% PAGE, and then transferred to Immobilon membranes, as indicated in Materials and Methods. Samples were analyzed for MARCKS phosphorylation at Ser-152/156 (pMARCKS) using a specific antibody that detects the phosphorylated state of these residues (A). Parallel membranes were analyzed by Western blotting using MARKCS (M-20) antibody to verify equal loading. Shown here is a representative autoluminogram. Graphed data (right) represents mean values (mean ± SEM; n = 9–10) of the level of MARCKS phosphorylation obtained from scanning densitometry. Control, Gray bars; insulin alone, striped bars; bradykinin without insulin pretreatment, white bars; bradykinin with insulin pretreatment, black bars. Samples were analyzed for PKD2 autophosphorylation at Ser-876 (pPKD2) using a specific antibody that detects the phosphorylated state of this residue (B). Parallel membranes were analyzed by Western blotting using PKCµ/PKD (C-20) antibody to verify equal loading. Shown here is a representative autoluminogram. Graphed data (right) represents mean values (mean ± SEM; n = 9) of the level of PKD2 autophosphorylation obtained from scanning densitometry. Control, Gray bars; insulin alone, striped bars; bradykinin without insulin pretreatment, white bars; bradykinin with insulin pretreatment, black bars. *, P < 0.05 and **, P < 0.01, statistical significances in the comparison of the effect of insulin with the corresponding bradykinin alone treatment.

The PI3-kinase/mTOR pathway mediates the insulin-induced potentiation of Ca²⁺ signaling and PtdIns(4,5)P₂ hydrolysis produced by G_qPCR agonists
To determine the mechanism by which insulin leads to enhanced PLC-mediated PtdIns(4,5)P₂ hydrolysis, production of Ins(1,4,5)P₃, and Ca²⁺ signaling in response to G_qPCR agonists, we determined the role of PI3-kinase/mTOR, a major insulin-induced pathway (36, 44). BxPc-3 cells were incubated with or without the selective inhibitors of PI3-kinase LY-294002 and wortmannin or with rapamycin, a selective inhibitor of mTOR in complex with the protein raptor (45, 57). Then, the treated cells were incubated or not with insulin and subsequently challenged with bradykinin. As seen in Fig. 6A, treatment of BxPc-3 cells with LY-294002, wortmannin, or rapamycin did not have any detectable effect on the increase in [Ca²⁺]_i induced by bradykinin but completely abolished the enhancement of Ca²⁺ signaling produced by previous exposure to insulin. We found that treatment with 10 nM rapamycin for only 10 min before the addition of insulin was sufficient to abolish insulin-induced enhancement of Ca²⁺ signaling (results not shown). In contrast, exposure to U0126, an inhibitor of MAPK kinase (MEK) 1, MEK2, and the closely related MEK5 (58), did not affect insulin-induced potentiation of bradykinin-elicited Ca²⁺ signaling.

View larger version (23K): [in this window] [in a new window]   FIG. 6. Treatment with LY-294002, wortmannin, or rapamycin prevents the insulin-induced potentiation of the increase in [Ca2+]i and Ins(1,4,5) synthesis stimulated by GPCR agonists. A, Effect of LY-294002, wortmannin, rapamycin, or U0126 on Ca2+ mobilization induced by bradykinin in BxPc-3 cells. BxPc-3 cells were pretreated with 10 µM LY-294002 (LY), 100 nM wortmannin (Wo), 10 nM rapamycin (Rapa), or 2.5 µM U0126 for 40 min before treating without (open bars) or with (filled bars) 10 ng/ml insulin for 5 min. Then, the cells were stimulated with 5 nM BK. [Ca2+]i was measured as described in Materials and Methods. Each panel represents four or more independent experiments; n = 12 for each condition. **, P < 0.01. B, Effect of LY-294002, wortmannin, or rapamycin on neurotensin (NT)-induced Ca2+ mobilization in PANC-1 cells. PANC-1 cells were pretreated with 10 µM LY-294002, 100 nM wortmannin, or 10 nM rapamycin for 30–45 min before stimulation with 5 nM neurotensin in the absence (open bars) or presence (filled bars) of a 5-min insulin pretreatment. Each panel represents at least four independent experiments; n = 12 for each condition. **, P < 0.01. C, Effect of LY-294002 and rapamycin on Ins(1,4,5)P3 synthesis stimulated by bradykinin in BxPc-3 cells. The cells were transiently transfected with a plasmid encoding GFP-PHD, as described above. The cultures were pretreated with 10 µM LY-294002 or 10 nM rapamycin for 30 min, perfused with RPMI (with HEPES) containing the inhibitors and 10 ng/ml insulin for 5 min before switching to perfusion fluid containing 5 nM bradykinin as described in Fig. 4. The intracellular distribution of GFP-PHD was monitored under fluorescence microscope (magnification, x400). The cells displayed were representative of 90% of the population of positive cells. D, Quantitative analysis of the relative change in plasma membrane (M) and cytosol (C) fluorescence intensity of individual cells treated with 5 nM bradykinin (white circles), bradykinin plus insulin pretreatment (black triangles), LY-294002 plus insulin pretreatment plus bradykinin (black squares), and rapamycin plus insulin pretreatment plus bradykinin (gray circles). The analysis was performed on 60 cells per experiment, with each experiment done at least in triplicate. *, P < 0.05, statistical significances in the comparison of the effect of the rapamycin and LY-294002 with the corresponding insulin pretreatments. There was no significant difference between the bradykinin alone, the LY-294002 plus insulin pretreatment plus bradykinin, and the rapamycin plus insulin pretreatment plus bradykinin groups.

To determine whether suppression of the PI3-kinase/Akt/mTOR also prevents the increase in the rate and extent of GPCR-induced hydrolysis of PtdIns(4,5)P₂ produced by exposure to insulin, we monitored the distribution of the biosensor for PtdIns(4,5)P₂ generation, GFP-PHD. LY-294002 or rapamycin delayed the redistribution of GFP-PHD from the plasma membrane to the cytosol after bradykinin stimulation in insulin-pretreated BxPc-3 cells (Fig. 6C). In the presence of these inhibitors, bradykinin-induced synthesis of Ins(1,4,5)P₃ in cells exposed to insulin was identical to that seen in control cells (Fig. 6D).

Insulin potentiates Ca²⁺ signaling in response to ANG II and vasopressin in normal intestinal epithelial IEC-18 cells
We next determined whether the ability of insulin to potentiate G_q signaling through an mTOR-dependent pathway could be demonstrated in normal as well as in cancer cells, using normal crypt-derived intestinal epithelial IEC-18 cells. Stimulation of these cells with either ANG II or vasopressin elicits Ca²⁺ signaling and PKC/PKD activation (46, 47, 48, 59, 60, 61), providing an additional model system to examine crosstalk between insulin receptor and GPCR signaling systems.

Similar to the results obtained with pancreatic cancer cells, exposure of IEC-18 cells to insulin (10 ng/ml for 5 min) markedly enhanced the peak and the rate of increase in [Ca²⁺]_i induced by a subsequent stimulation with either ANG II (Fig. 7A) or vasopressin (Fig. 7B). Treatment with LY-294002 completely abrogated and rapamycin markedly attenuated the potentiation of Ca²⁺ signaling induced by insulin in IEC-18 cells challenged with the G_qPCR agonist vasopressin (Fig. 7C). These results indicate that the crosstalk between insulin and G_qPCR agonists identified in this study is not restricted to cancer cells but can be demonstrated in nontumorigenic cell model systems.

View larger version (13K): [in this window] [in a new window]   FIG. 7. Exposure to insulin enhances the [Ca2+]i increase induced by ANG II or vasopressin in IEC-18 cells through an mTOR-dependent pathway. A and B, Insulin enhances the increase in [Ca2+]i induced by ANG II or vasopressin (VP) in IEC-18 cells. IEC-18 cells were loaded with fura 2-AME, and [Ca2+]i was measured as described in Materials and Methods. Left, Tracings of [Ca2+]i in response to 50 nM ANG II (A) or to 50 nM vasopressin (B) in IEC-18 cells treated with (thick traces) and without (thin traces) 10 ng/ml insulin for 5 min. Right, Peak, The maximum increment in [Ca2+]i in response to ANG II (A) or vasopressin (B) without (open bars) or with (filled bars) insulin pretreatment. Rate, The slope of the trace of increase in [Ca2+]i after the addition of ANG II (A) or vasopressin (B) in the absence (open bars) or presence (filled bars) of insulin pretreatment. The units represent the increment in [Ca2+]i during the first 10 sec from the moment that [Ca2+]i started to increase. The panels represent at least four independent experiments for each agonist (n = 16 for ANG II alone, n = 20 for ANG II after insulin pretreatment; n = 12 for vasopressin alone and n = 16 for vasopressin after insulin pretreatment). C, Effect of LY-294002 (LY) and rapamycin (Rapa) on [Ca2+]i increase stimulated by vasopressin in IEC-18 cells treated without (open bars) or with (filled bars) insulin. IEC-18 cells were pretreated with 10 µM LY-294002 or 10 nM rapamycin for 30 min before stimulation with 50 nM vasopressin in cells treated with or without insulin. Each panel represents at least three independent experiments (n = 12 for each condition). *, P < 0.05; **, P < 0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We produced several lines of evidence indicating that insulin enhances PLC-mediated PtdIns(4,5)P₂ hydrolysis, production of Ins(1,4,5)P₃, Ca²⁺ mobilization from internal stores, and DAG-stimulated PKC elicited by G_qPCRs: 1) cell exposure to insulin enhanced Ca²⁺ signaling in response to activation of multiple G_qPCRs, including those recognizing the biologically active peptides ANG II, bradykinin, bombesin, neurotensin, and vasopressin; 2) insulin did not increase Ca²⁺ signaling by thapsigargin, an agent that bypasses receptor-mediated pathways and directly discharges internal pools; 3) insulin did not augment Ca²⁺ signaling in response to LPA, a GPCR agonist that increases [Ca²⁺]_i through a PTx-sensitive G_i signaling pathway rather than via G_q in pancreatic cancer cells; 4) using a fusion protein between GFP and the PH domain of PLC1, a sensor of PLC-mediated Ins(1,4,5)P₃ synthesis (49, 53, 54), we demonstrated that exposure to insulin increased the rate and extent of Ins(1,4,5)P₃ production induced by bradykinin; and 5) cell exposure to insulin enhanced bradykinin-induced MARCKS phosphorylation and PKD2 activation. Collectively, these results provide strong support for the notion that exposure to insulin potentiates early signaling steps in G_qPCR action.

As a first step to elucidate the mechanism(s) by which insulin potentiates G_qPCR signaling, we attempted to identify the signaling pathway involved in insulin-induced potentiation of GPCR signaling. A major pathway stimulated by activation of the insulin receptor is the PI3-kinase/Akt/mTOR module. Recent evidence indicates that mTOR is a major point of convergence of signals from mitogenic growth factors, nutrients, cellular energy levels, and stress conditions to stimulate protein synthesis and cell growth (45). Interestingly, two distinct TOR complexes, TORC1 and TORC2, have been characterized. TORC1 is sensitive to rapamycin and is responsible for phosphorylation of p70S6K and 4EBP1. In contrast, TORC2 is not inhibited by rapamycin (45, 57). Our results show that short-term treatment with either rapamycin or the PI3-kinase inhibitors LY-294002 and wortmannin completely abrogated the ability of insulin to increase the rate and magnitude of Ca²⁺ signaling and production of Ins(1,4,5)P₃ in response to GPCR activation in pancreatic cancer cells. Our results also show that treatment with rapamycin or LY-294002 prevented the ability of insulin to increase Ca²⁺ signaling in response to GPCR activation in normal intestinal epithelial IEC-18 cells. These results indicate, for the first time, that insulin potentiates G_q/PLC signaling through a TORC1-dependent pathway in a variety of cell model systems.

Although our results show that cell exposure to insulin markedly enhanced subsequent GPCR-induced Ca²⁺ signaling, we also found that the addition of these ligands in reverse order, i.e. addition of insulin after GPCR agonists, did not produce any additional increase in [Ca²⁺]_i. In this context, recent studies demonstrated that an elevation in [Ca²⁺]_i negatively regulates mTOR through tuberous sclerosis complex 2 phosphorylation via the AMP-activated protein kinase pathway (63, 64, 65). Thus, an increase in the activity of the Ca²⁺/AMP-activated protein kinase pathway could act as a feedback mechanism that prevents excessive (potentially toxic) G_qPCR-induced increases in [Ca²⁺]_i in cells receiving a concomitant insulin signal.

It is increasingly recognized that the crosstalk between insulin and GPCR signaling is critical for the regulation of multiple physiological functions and underlies the pathogenesis of important diseases, including cancer (for references, see introductory section). In this context, the incidence of obesity, metabolic syndrome, hypertension, and type II diabetes mellitus, characterized by insulin resistance and compensatory hyperinsulinemia, is reaching alarming rates in the developed world (10, 11, 66, 67). Our results, identifying a crosstalk mechanism by which insulin potentiates Ca²⁺ mobilization from internal stores and PKC/PKD activation in response to G_qPCR activation, have, therefore, important implications. The magnitude, rate, and duration of increases in [Ca²⁺]_i are increasingly recognized as encoding critical information for a variety of biological processes, including secretion, differential gene expression, and cell proliferation (42). Given the close relationship between the endocrine and exocrine pancreas through the insuloacinar portal system (68) and the local production of ANG II in the pancreas (69), the crosstalk between insulin and G_qPCRs is of especial significance for acinar and ductal pancreatic cells, but it is likely to be of importance for other cell types in the organism, in particular in states characterized by hyperinsulinemia. In view of the importance of gastrointestinal hormones in the function of intestinal epithelial cells (70) and the association between hyperinsulinemia and colon cancer (16, 17, 18), it is of interest that insulin also enhanced Ca²⁺ signaling in response to G_qPCR agonists in intestinal epithelial cells.

In conclusion, we propose that the potentiation of GPCR signaling by insulin through a TORC1-dependent pathway provides a mechanism by which insulin enhances the responsiveness of many cells and tissues to G_qPCR agonists, including G_qPCR-mediated autocrine and paracrine loops in cancer cells.

	Acknowledgments

 
We gratefully acknowledge support from the Morphology/Imaging Core of the Center for Ulcer Research and Education: Digestive Diseases Research Core Center (National Institute of Diabetes and Digestive and Kidney Diseases Grant 5 P30 DK41301). We thank Drs. A. Lunn and R. Waldron for critical reading of this manuscript.

	Footnotes

 
E.R. is the Ronald S. Hirshberg Professor of Pancreatic Cancer Research. This work was supported by National Institutes of Health Grants RO1 DK 56930, RO1 DK 55003, and P30 DK41301. O.R. is a recipient of a National Cancer Institute Mentored Career Development Award K01CA097956.

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 22, 2007

Abbreviations: ACE, Angiotensin-converting enzyme; ANG II, angiotensin II; [Ca²⁺]_i, intracellular Ca²⁺ concentration; DAG, diacylglycerol; FBS, fetal bovine serum; fura 2-AME, fura 2-tetra-acetoxy methyl ester; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; G_qPCR, G_q protein-coupled receptor; Ins(1,4,5)P₃, inositol 1,4,5-trisphosphate; LPA, lysophosphatidic acid; MARCKS, myristoylated alanine-rich C kinase substrate; MEK, MAPK kinase; PHD, pleckstrin homology domain; PI3K, phosphatidylinositol 3 kinase; PLC, phospholipase C; PKC, protein kinase C; PKD, protein kinase D; PtdIns(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; PTx, pertussis toxin.

Received December 19, 2006.

Accepted for publication March 15, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Velloso LA, Folli F, Perego L, Saad MJ 2006 The multi-faceted cross-talk between the insulin and angiotensin II signaling systems. Diabetes Metab Res Rev 22:98–107[CrossRef][Medline]
2. Kakoki M, Kizer CM, Yi X, Takahashi N, Kim HS, Bagnell CR, Edgell CJ, Maeda N, Jennette JC, Smithies O 2006 Senescence-associated phenotypes in Akita diabetic mice are enhanced by absence of bradykinin B2 receptors. J Clin Invest 116:1302–1309[CrossRef][Medline]
3. Kakoki M, Takahashi N, Jennette JC, Smithies O 2004 Diabetic nephropathy is markedly enhanced in mice lacking the bradykinin B2 receptor. Proc Natl Acad Sci USA 101:13302–13305[Abstract/Free Full Text]
4. Sowers JR 2004 Insulin resistance and hypertension. Am J Physiol Heart Circ Physiol 286:H1597–H1602
5. Aguilar D, Solomon SD 2006 ACE inhibitors and angiotensin receptor antagonists and the incidence of new-onset diabetes mellitus: an emerging theme. Drugs 66:1169–1177[CrossRef][Medline]
6. Haffner SM 2006 Risk constellations in patients with the metabolic syndrome: epidemiology, diagnosis, and treatment patterns. Am J Med 119:S3–S9
7. Hansson L, Lindholm LH, Niskanen L, Lanke J, Hedner T, Niklason A, Luomanmaki K, Dahlof B, de Faire U, Morlin C 1999 Effect of angiotensin-converting-enzyme inhibition compared with conventional therapy on cardiovascular morbidity and mortality in hypertension: the Captopril Prevention Project (CAPPP) randomised trial. Lancet 353:611–616[CrossRef][Medline]
8. Hunyady L, Catt KJ 2006 Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II. Mol Endocrinol 20:953–970[Abstract/Free Full Text]
9. Coughlin SS, Calle EE, Teras LR, Petrelli J, Thun MJ 2004 Diabetes mellitus as a predictor of cancer mortality in a large cohort of US adults. Am J Epidemiol 159:1160–1167[Abstract/Free Full Text]
10. Calle EE, Kaaks R 2004 Overweight, obesity and cancer: epidemiological evidence and proposed mechanisms. Nat Rev Cancer 4:579–591[CrossRef][Medline]
11. Calle EE, Thun MJ 2004 Obesity and cancer. Oncogene 23:6365–6378[CrossRef][Medline]
12. Jee SH, Ohrr H, Sull JW, Yun JE, Ji M, Samet JM 2005 Fasting serum glucose level and cancer risk in Korean men and women. JAMA 293:194–202[Abstract/Free Full Text]
13. Chari ST, Leibson CL, Rabe KG, Ransom J, de Andrade M, Petersen GM 2005 Probability of pancreatic cancer following diabetes: a population-based study. Gastroenterology 129:504–511[CrossRef][Medline]
14. Huxley R, Ansary-Moghaddam A, Berrington de Gonzalez A, Barzi F, Woodward M 2005 Type-II diabetes and pancreatic cancer: a meta-analysis of 36 studies. Br J Cancer 92:2076–2083[CrossRef][Medline]
15. Larsson SC, Bergkvist L, Wolk A 2006 Consumption of sugar and sugar-sweetened foods and the risk of pancreatic cancer in a prospective study. Am J Clin Nutr 84:1171–1176[Abstract/Free Full Text]
16. Bowers K, Albanes D, Limburg P, Pietinen P, Taylor PR, Virtamo J, Stolzenberg-Solomon R 2006 A prospective study of anthropometric and clinical measurements associated with insulin resistance syndrome and colorectal cancer in male smokers. Am J Epidemiol 164:652–664[Abstract/Free Full Text]
17. Elwing JE, Gao F, Davidson NO, Early DS 2006 Type 2 diabetes mellitus: the impact on colorectal adenoma risk in women. Am J Gastroenterol 101:1866–1871[CrossRef][Medline]
18. Seow A, Yuan JM, Koh WP, Lee HP, Yu MC 2006 Diabetes mellitus and risk of colorectal cancer in the Singapore Chinese Health Study. J Natl Cancer Inst 98:135–138[Abstract/Free Full Text]
19. Hammarsten J, Hogstedt B 2005 Hyperinsulinaemia: a prospective risk factor for lethal clinical prostate cancer. Eur J Cancer 41:2887–2895[CrossRef][Medline]
20. Lever AF, Hole DJ, Gillis CR, McCallum IR, McInnes GT, MacKinnon PL, Meredith PA, Murray LS, Reid JL, Robertson JW 1998 Do inhibitors of angiotensin-I-converting enzyme protect against risk of cancer? Lancet 352:179–184[CrossRef][Medline]
21. Uemura H, Ishiguro H, Nagashima Y, Sasaki T, Nakaigawa N, Hasumi H, Kato S, Kubota Y 2005 Antiproliferative activity of angiotensin II receptor blocker through cross-talk between stromal and epithelial prostate cancer cells. Mol Cancer Ther 4:1699–1709[Abstract/Free Full Text]
22. Ryder NM, Guha S, Hines OJ, Reber HA, Rozengurt E 2001 G protein-coupled receptor signaling in human ductal pancreatic cancer cells: neurotensin responsiveness and mitogenic stimulation. J Cell Physiol 186:53–64[CrossRef][Medline]
23. Guha S, Rey O, Rozengurt E 2002 Neurotensin induces protein kinase C-dependent protein kinase D activation and DNA synthesis in human pancreatic carcinoma cell line PANC-1. Cancer Res 62:1632–1640[Abstract/Free Full Text]
24. Rey O, Yuan J, Rozengurt E 2003 Intracellular redistribution of protein kinase D2 in response to G- protein-coupled receptor agonists. Biochem Biophys Res Commun 302:817–824[CrossRef][Medline]
25. Guha S, Lunn JA, Santiskulvong C, Rozengurt E 2003 Neurotensin stimulates protein kinase C-dependent mitogenic signaling in human pancreatic carcinoma cell line PANC-1. Cancer Res 63:2379–2387[Abstract/Free Full Text]
26. Kisfalvi K, Guha S, Rozengurt E 2005 Neurotensin and EGF induce synergistic stimulation of DNA synthesis by increasing the duration of ERK signaling in ductal pancreatic cancer cells. J Cell Physiol 202:880–890[CrossRef][Medline]
27. Guha S, Eibl G, Kisfalvi K, Fan RS, Burdick M, Reber H, Hines OJ, Strieter R, Rozengurt E 2005 Broad-spectrum G protein-coupled receptor antagonist, [D-Arg1,D-Trp5,7,9,Leu11]SP: a dual inhibitor of growth and angiogenesis in pancreatic cancer. Cancer Res 65:2738–2745[Abstract/Free Full Text]
28. Young SH, Rozengurt E 2006 Qdot nanocrystal conjugates conjugated to bombesin or ANG II label the cognate G protein-coupled receptor in living cells. Am J Physiol Cell Physiol 290:C728–C732
29. Rozengurt E 1992 Growth factors and cell proliferation. Curr Opin Cell Biol 4:161–165[CrossRef][Medline]
30. Rozengurt E 1998 Signal transduction pathways in the mitogenic response to G protein-coupled neuropeptide receptor agonists. J Cell Physiol 177:507–517[CrossRef][Medline]
31. Gutkind JS 1998 Cell growth control by G protein-coupled receptors: from signal transduction to signal integration. Oncogene 17:1331–1342[CrossRef][Medline]
32. Rozengurt E 1999 Autocrine loops, signal transduction, and cell cycle abnormalities in the molecular biology of lung cancer. Curr Opin Oncol 11:116–122[CrossRef][Medline]
33. Rozengurt E, Walsh JH 2001 Gastrin, CCK, signaling, and cancer. Annu Rev Physiol 63:49–76[CrossRef][Medline]
34. Heasley LE 2001 Autocrine and paracrine signaling through neuropeptide receptors in human cancer. Oncogene 20:1563–1569[CrossRef][Medline]
35. Rozengurt E 2002 Neuropeptides as growth factors for normal and cancer cells. Trends Endocrinol Metabol 13:128–134[CrossRef][Medline]
36. Saltiel AR, Pessin JE 2002 Insulin signaling pathways in time and space. Trends Cell Biol 12:65–71[CrossRef][Medline]
37. Hur EM, Kim KT 2002 G protein-coupled receptor signalling and cross-talk: achieving rapidity and specificity. Cell Signal 14:397–405[CrossRef][Medline]
38. Pawson T 2004 Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116:191–203[CrossRef][Medline]
39. Rhee SG 2001 Regulation of phosphoinositide-specific phospholipase C. Annu Rev Biochem 70:281–312[CrossRef][Medline]
40. Mikoshiba K 1997 The InsP3 receptor and intracellular Ca²⁺ signaling. Curr Opin Neurobiol 7:339–345[CrossRef][Medline]
41. Exton JH 1996 Regulation of phosphoinositide phospholipases by hormones, neurotransmitters, and other agonists linked to G proteins. Annu Rev Pharmacol Toxicol 36:481–509[CrossRef][Medline]
42. Berridge MJ, Lipp P, Bootman MD 2000 The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1:11–21[CrossRef][Medline]
43. Rozengurt E, Rey O, Waldron RT 2005 Protein kinase D signaling. J Biol Chem 280:13205–13208[Free Full Text]
44. Um SH, D’Alessio D, Thomas G 2006 Nutrient overload, insulin resistance, and ribosomal protein S6 kinase 1, S6K1. Cell Metab 3:393–402[CrossRef][Medline]
45. Wullschleger S, Loewith R, Hall MN 2006 TOR signaling in growth and metabolism. Cell 124:471–484[CrossRef][Medline]
46. Chiu T, Rozengurt E 2001 PKD in intestinal epithelial cells: rapid activation by phorbol esters, LPA, and angiotensin through PKC. Am J Physiol Cell Physiol 280:C929–C942
47. Chiu T, Wu SS, Santiskulvong C, Tangkijvanich P, Yee Jr HF, Rozengurt E 2002 Vasopressin-mediated mitogenic signaling in intestinal epithelial cells. Am J Physiol Cell Physiol 282:C434–C450
48. Chiu T, Santiskulvong C, Rozengurt E 2003 ANG II stimulates PKC-dependent ERK activation, DNA synthesis, and cell division in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 285:G1–G11
49. Rey O, Young SH, Yuan J, Slice L, Rozengurt E 2005 Amino acid-stimulated Ca²⁺ oscillations produced by the Ca²⁺-sensing receptor are mediated by a phospholipase C/inositol 1,4,5-trisphosphate-independent pathway that requires G12, Rho, filamin-A, and the actin cytoskeleton. J Biol Chem 280:22875–22882[Abstract/Free Full Text]
50. Nigro J, Osman N, Dart AM, Little PJ 2006 Insulin resistance and atherosclerosis. Endocr Rev 27:242–259[Abstract/Free Full Text]
51. Hotz HG, Reber HA, Hotz B, Yu T, Foitzik T, Buhr HJ, Cortina G, Hines OJ 2003 An orthotopic nude mouse model for evaluating pathophysiology and therapy of pancreatic cancer. Pancreas 26:e89–e98
52. Thastrup O, Cullen PJ, Drøbak BK, Hanley MR, Dawson AP 1990 Thapsigargin, a tumor promoter, discharges intracellular Ca²⁺ stores by specific inhibition of the endoplasmic reticulum Ca²⁺-ATPase. Proc Natl Acad Sci USA 87:2466–2470[Abstract/Free Full Text]
53. Nash MS, Young KW, Challiss RA, Nahorski SR 2001 Intracellular signalling. Receptor-specific messenger oscillations. Nature 413:381–382[CrossRef][Medline]
54. van der Wal J, Habets R, Varnai P, Balla T, Jalink K 2001 Monitoring agonist-induced phospholipase C activation in live cells by fluorescence resonance energy transfer. J Biol Chem 276:15337–15344[Abstract/Free Full Text]
55. Blackshear PJ 1993 The MARCKS family of cellular protein kinase C substrates. J Biol Chem 268:1501–1504[Free Full Text]
56. Herget T, Oehrlein SA, Pappin DJ, Rozengurt E, Parker PJ 1995 The myristoylated alanine-rich C-kinase substrate (MARCKS) is sequentially phosphorylated by conventional, novel and atypical isotypes of protein kinase C. Eur J Biochem 233:448–457[Medline]
57. Inoki K, Guan KL 2006 Complexity of the TOR signaling network. Trends Cell Biol 16:206–212[CrossRef][Medline]
58. Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, Trzaskos JM 1998 Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273:18623–18632[Abstract/Free Full Text]
59. Chiu T, Santiskulvong C, Rozengurt E 2005 EGF receptor transactivation mediates ANG II-stimulated mitogenesis in intestinal epithelial cells through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway. Am J Physiol Gastrointest Liver Physiol 288:G182–G194
60. Rey O, Zhukova E, Sinnett-Smith J, Rozengurt E 2003 Vasopressin-induced intracellular redistribution of protein kinase D in intestinal epithelial cells. J Cell Physiol 196:483–492[CrossRef][Medline]
61. Wu SS, Chiu T, Rozengurt E 2002 ANG II and LPA induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells: role of Ca²⁺, PKC, and Rho kinase. Am J Physiol Cell Physiol 282:C1432–C1444
62. Wei Y, Sowers JR, Nistala R, Gong H, Uptergrove GM-E, Clark SE, Morris EM, Szary N, Manrique C, Stump CS 2006 Angiotensin II-induced NADPH oxidase activation impairs insulin signaling in skeletal muscle cells. J Biol Chem 281:35137–35146[Abstract/Free Full Text]
63. Woods A, Dickerson K, Heath R, Hong SP, Momcilovic M, Johnstone SR, Carlson M, Carling D 2005 Ca²⁺/calmodulin-dependent protein kinase kinase-ß acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab 2:21–33[CrossRef][Medline]
64. Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM, Frenguelli BG, Hardie DG 2005 Calmodulin-dependent protein kinase kinase-ß is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2:9–19[CrossRef][Medline]
65. Hurley RL, Anderson KA, Franzone JM, Kemp BE, Means AR, Witters LA 2005 The Ca²⁺/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases. J Biol Chem 280:29060–29066[Abstract/Free Full Text]
66. Flier JS 2004 Obesity wars: molecular progress confronts an expanding epidemic. Cell 116:337–350[CrossRef][Medline]
67. Muoio DM, Newgard CB 2006 Obesity-related derangements in metabolic regulation. Annu Rev Biochem 75:367–401[CrossRef][Medline]
68. Lee KY, Zhou L, Ren XS, Chang TM, Chey WY 1990 An important role of endogenous insulin on exocrine pancreatic secretion in rats. Am J Physiol 258:G268–G274
69. Fleming I, Kohlstedt K, Busse R 2006 The tissue renin-angiotensin system and intracellular signalling. Curr Opin Nephrol Hypertens 15:8–13[Medline]
70. Wang J, Cortina G, Wu SV, Tran R, Cho JH, Tsai MJ, Bailey TJ, Jamrich M, Ament ME, Treem WR, Hill ID, Vargas JH, Gershman G, Farmer DG, Reyen L, Martin MG 2006 Mutant neurogenin-3 in congenital malabsorptive diarrhea. N Engl J Med 355:270–280[Abstract/Free Full Text]

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