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Regulation of Glucagon-Like Peptide-1 Receptor and Calcium-Sensing Receptor Signaling by L-Histidine

Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Colin A. Leech, Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, 50 Blossom Street, Boston, Massachusetts 02114. E-mail: leech{at}helix.mgh.harvard.edu.

	Abstract

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Receptor-specific agonists of the extracellular calcium-sensing receptor (CaSR) potentiate glucose-induced insulin secretion, an effect similar to that of glucagon-like peptide-1 (GLP-1). We have sequenced the full open reading frame of the CaSR from rat insulinoma (INS-1) cells and find that the predicted amino acid sequence of the receptor is identical with that of the receptor from the parathyroid gland. This receptor couples to both Gq/11 and Gi/o, and this dual coupling may partly explain the varying effects of nonspecific agonists on secretion reported previously. L-Histidine (L-His) increases the sensitivity of the CaSR to extracellular Ca²⁺ and potentiates glucose-dependent insulin secretion from INS-1 cells. This potentiation is partially inhibited at low extracellular [Ca²⁺] where the CaSR is ineffective. Coexpression of the CaSR and GLP-1 receptor (GLP-1R) produces a pertussis toxin-sensitive inhibition of GLP-1-induced cAMP production in response to elevated extracellular [Ca²⁺]. However, L-His potentiates cAMP response element reporter activity in INS-1 cells and in human embryonic kidney-293 cells expressing either the GLP-1R alone or the CaSR and GLP-1R. INS-1 cells express the RNA for the CaSR at a lower level than that for the GLP-1R. This difference in expression level of the receptors may explain the potentiation of insulin secretion by L-His despite coupling of the CaSR to Gi/o. In conclusion, L-His can potentiate both GLP-1R- and CaSR-activated signaling pathways, and these effects may play a role in the potentiation of glucose-induced insulin secretion in response to meals containing protein in addition to carbohydrates and fat.

	Introduction

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THE PRESENCE OF a plasma membrane cationic binding site in ß-cells was proposed from studies on the inhibitory effects of lanthanides on insulin secretion performed in 1980 (1). The existence of a cell surface receptor for divalent cations in neonatal rat islets was then suggested in 1995 (2). Partial sequence data reported in subsequent studies have shown the expression of the parathyroid extracellular Ca²⁺-sensing receptor (CaSR) in human islets (3) and human insulinoma cells (4) whose amino acid sequence is identical with the CaSR cloned from human kidney (5). Partial sequence data for the CaSR from rat islets (6) and rat pancreas (7), and the full open reading frame sequence from rat insulinoma (INS-1) cell cDNA (reported here), match the sequence of rat neuronal (8) and kidney (9) CaSRs. However, there is not yet a consensus on the importance of this G protein-coupled receptor in the regulation of insulin secretion (3, 6, 10, 11).

Experiments using human islets and human clonal cell lines in which extracellular [Ca²⁺] ([Ca²⁺]_o) was elevated showed that insulin secretion was transiently increased (3, 12) followed either by a return to basal levels (12) or by inhibition of secretion (3). These complex effects on insulin secretion may reflect the coupling of the CaSR to both Gq/11 and Gi/o (13) that, respectively, have stimulatory and inhibitory effects on secretion. Data showing that nonspecific CaSR agonists inhibit insulin secretion are also likely to reflect receptor-independent inhibitory effects. For example, the CaSR agonist neomycin, reported to have no effect (11) or to inhibit insulin secretion (10), inhibits voltage-dependent calcium channels (14), intracellular Ca²⁺ ([Ca²⁺]_i) release from ryanodine-sensitive stores (15), and phospholipase C (PLC) through binding to phosphatidylinositol (4, 5)-bisphosphate (16). All these CaSR-independent effects of neomycin will inhibit insulin secretion independently of its activity as an agonist of the CaSR and complicate the interpretation of its effects on secretion. However, experiments using a relatively specific CaSR agonist, the phenylalkylamine R-467, showed a potentiation of glucose-induced insulin secretion from mouse islets and ßHC9 insulinoma cells (11).

It is established that the CaSR is sensitive not only to extracellular [Ca²⁺], but also to amino acids at concentrations found postprandially in the circulation (17). This amino acid sensitivity of the CaSR provides a physiological basis for its expression in ß-cells as a modulator of nutrient-induced insulin secretion. Herein we present data showing that the amino acid L-His potentiates glucose-induced insulin secretion and propose that this effect is mediated, at least in part, by increased activation of the CaSR. The signaling pathways activated by the CaSR in the parathyroid gland are mediated by Gq/11 and Gi/o (13, 18). However, data obtained from studies on ß-cells suggest that Gq/11, PLC, and inositol trisphosphate production and cAMP production are not affected by the CaSR (3, 10, 11). These findings raise the possibility that ß-cells may express a unique form of the CaSR and thereby representing a potential target for the treatment of diabetes. This possibility led us to isolate and to sequence the full open reading frame of the CaSR cDNA from INS-1 cells.

Our hypothesis is that the physiological role of the CaSR in ß-cells is to sense increases in amino acid concentrations postprandially and to potentiate glucose-induced insulin secretion. We also show that the amino acids L-His and L-leucine (L-Leu) [and its nonmetabolizable analog, 2-amino-2-norbornane-carboxylic acid (BCH)] potentiate cAMP reporter activity induced by GLP-1. These receptor-mediated effects may contribute to the left-shift of the glucose dependence of insulin secretion observed previously in the presence of physiological amino acid levels (19).

	Materials and Methods

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Materials
All chemical reagents were obtained from Sigma (St. Louis, MO), unless otherwise stated.

Insulin secretion assays from INS-1 cells
INS-1 cells grown in RPMI 1640 culture medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 5 µl/liter ß-mercaptoethanol (Life Technologies, Inc., Carlsbad, CA) were split into 12-well plates for insulin secretion assays. Various concentrations of glucose and amino acids (detailed in results) were added to a standard extracellular solution (SES) containing: 138 mM NaCl, 5.6 mM KCl, 2.6 mM MgCl₂, 1.2 mM MgCl₂, 10 mM HEPES (pH adjusted to 7.4 with NaOH, approximately 4 mM). Human serum albumin (0.05%) was also added to the SES for insulin secretion assays and GLP-1 stimulation experiments. Plates of INS-1 cells were washed with SES containing 2 mM glucose before the addition of experimental test solutions and incubated at 37 C for 45 min in both wash and test solutions. Aliquots of the bathing solution were then collected and assayed for insulin content using a Linco (St. Charles, MO) RIA kit.

Perforated patch voltage clamp
Cells were plated onto glass coverslips coated with concanavalin A and placed in a temperature-controlled chamber held at 32 C. The bath solution was refreshed or exchanged using a multivalve perfusion system (Warner, Hamden, CT; VC-6) with solutions passing through an in-line heater (Warner SH-27B/TC-324B) to maintain bath temperature. The bath solution was 2 mM glucose SES and Ca²⁺ currents were recorded using a Cs⁺ pipette solution (to block outward K⁺ currents) containing: 75 mM Cs₂SO₄, 25 mM K₂SO₄, 10 mM NaCl, 2.6 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES (pH adjusted to 7.4 with KOH, approximately 2 mM). A stock solution of nystatin (60 mg/ml in dimethylsulfoxide) was made fresh daily and diluted into pipette solution to a final concentration of 240 µg/ml. This final nystatin solution was used within 2 h.

Patch pipettes were pulled from borosilicate glass capillaries, tip-dipped in nystatin-free pipette solution and then back-filled with nystatin-containing solution. Pipettes were connected to a Heka (Southboro, MA) EPC-9 amplifier controlled using Pulse (Southboro, MA) version 8.53 software. Evoked currents were stored on a personal computer and analyzed using PulseFit (Southboro, MA) software.

Transient transfection of cells with CaSR, GLP-1R expression vectors, and luciferase reporters
Human embryonic kidney (HEK)-293 cells were grown in 10-cm culture dishes to 80% confluence before transient transfections of plasmid vectors using Lipofectamine (Invitrogen). Cells were transfected for 6 h in serum-free DMEM (Life Technologies) containing 20 µl Lipofectamine (Life Technologies), 1 µg of CaSR- or GLP-1R-pcDNA3.1 expression vector, and 230 ng of pCRE-Luc reporter (Stratagene, La Jolla, CA) for cAMP assays. Transfected cells were then washed with normal culture medium (DMEM + 10% FBS + 1% penicillin/streptomycin) and incubated overnight before splitting into 24-well plates. After a further 24 h, cells were washed with serum-free DMEM containing 0.05% human serum albumin and various agonists added for 4 h. The luciferase activity was measured using the Promega (Madison, WI) luciferase system in a Berthold (Bad Wildbad, Germany) Autolumat Plus luminometer or a Victor 2 (Wallac, Turku, Finland) plate reader.

INS-1 cells were grown to about 80% confluence and transfected in 10-cm dishes using 20 µl of Lipofectamine 2000 and 500 ng of pCRE-Luc reporter in serum-free RPMI 1640 medium overnight. Some experiments were performed using dual luciferase assays when cells were cotransfected with 500 ng of pCRE-Luc and 2 ng of phRL-CMV Renilla luciferase reporter (Promega). Transfected INS-1 cells were washed with normal culture medium (RPMI 1640 + 10% FBS + 1% penicillin/streptomycin + 5 µl/liter ß-mercaptoethanol) and treated as described above. Luciferase activity was measured using Promega single or dual luciferase assay systems.

Quantitative PCR (QPCR)
QPCR experiments were performed using a Cepheid SmartCycler system with cDNA derived from INS-1 cells. Relative quantification of CaSR and GLP-1 receptor (GLP-1R) cDNA levels was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference. Primers and Taqman probes were designed using Beacon Designer 2 software (Premier Biosoft, Palo Alto, CA) for rat-specific sequences of GAPDH, CaSR and GLP-1R. The sequences of the primers and probes were: GAPDH sense primer, TGGTC TACAT GTTCC AGTAT GACT; antisense primer, CCATT TGATG TTAGC GGGAT CTC; probe, CCACG GCAAG TTCAA CGGCA CAGT; CaSR sense primer, CTGCT TTGAG TGTGT GGAGT GT; antisense primer, GGTTC TCATT GGACC AGAAG TCAT; probe, CGGGC ACTTG TCACA GGCAC TCGC; GLP-1R sense primer, CAGCA GCATG AAACC CCTCA A; antisense primer, GCAGC AAGGA CCATC AGGAA G; probe, TGTCC CACCA GCAGC GTCAG CAGT. To test for equal amplification efficiencies of the target and reference reactions, the difference in threshold cycle (Ct) between the receptor and GAPDH was measured at different cDNA dilutions [1x, 0.1x, and 0.01x (20)]. The Ct values did not change with cDNA dilution (data not shown) indicating that the amplification efficiencies of the reactions are equal and allowing relative quantification. Test experiments were performed in 25-µl reaction tubes using FastStart Polymerase (Roche, Indianapolis, IN) at two cDNA concentrations (1x and 0.1x) with an initial step at 95 C for 240 sec followed by 40 cycles of 95 C, 30 sec and 58 C, 40 sec. Fluorescence was measured during the 58 C annealing and extension phase of each cycle.

Statistical analysis
Statistical analysis was performed with Origin (version 7.0) software (OriginLab, Northampton, MA) using ANOVA. All values are plotted as the mean and SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The CaSR-specific agonist R-467 potentiates glucose-induced insulin secretion only at elevated glucose levels (11). A variety of L-amino acids act as natural agonists for the CaSR (17) and CaSR-mediated effects of amino acids on insulin secretion would therefore be expected to show a similar glucose dependence to R-467. L-His produces the largest increase in the sensitivity of the CaSR to Ca²⁺ (17), and therefore we focused our initial experiments using L-His. Figure 1A shows that insulin secretion from INS-1 cells was significantly increased by raising glucose from 2–10 mM (P < 0.001, Fig. 1A). Addition of 5 mM L-His at 10 mM glucose, but not at 2 mM glucose, significantly potentiated insulin secretion (P < 0.001, Fig. 1A). Addition of 5 mM of the enantiomer D-His had no significant effect at either glucose concentration (Fig. 1A). These effects of L- and D-His on secretion are consistent with the stereospecificity of the CaSR to L- vs. D-amino acids (17).

View larger version (22K): [in this window] [in a new window]   FIG. 1. A, L-His (LH) but not D-His (DH) potentiates glucose-dependent insulin secretion from INS-1 cells. Results shown are normalized to secretion at 2 mM glucose and are averaged from duplicate assays of six samples from 22 (glucose alone), 15 (LH), and seven (DH) experiments. B, Leucine (L) and its nonmetabolizable analog BCH (B) are ineffective on the CaSR but significantly potentiate secretion at both 2 mM and 10 mM glucose. Data are averaged from duplicate assays of six samples from three experiments.

The effects of amino acids on the CaSR are inhibited at low (<1 mM) extracellular [Ca²⁺] ([Ca²⁺]_o) (17), and hence CaSR-mediated effects of L-His on insulin secretion are predicted to be inhibited at low [Ca²⁺]_o. However, decreasing [Ca²⁺]_o has receptor-independent effects, including a reduction of Ca²⁺ current amplitude that plays an important physiological role in triggering insulin secretion (22). We therefore measured Ca²⁺ currents (I_Ca) using perforated patch voltage clamp at both normal (2.6 mM) and low (0.5 mM) [Ca²⁺]_o (Fig. 2A). Having measured the reduction in I_Ca under low [Ca²⁺]_o conditions, we experimentally determined a concentration of Cd²⁺ (10 µM) that produced a similar degree of inhibition of I_Ca (Fig. 2A). The use of Cd²⁺ acts a control for the decrease in I_Ca in experiments where [Ca²⁺]_o was decreased to 0.5 mM while maintaining activity of the CaSR. Insulin secretion experiments were then performed in which [Ca²⁺]_o was decreased from 2.6 mM to 0.5 mM or where 10 µM Cd²⁺ was added to the 2.6 mM [Ca²⁺]_o solutions (Fig. 2B). Control secretory responses in 2.6 mM [Ca²⁺]_o showed glucose-stimulated insulin secretion that was potentiated by 56% at 10 mM glucose by the addition of 5 mM L-His. No significant effect of L-His was seen at 2 mM glucose. Decreasing [Ca²⁺]_o from 2.6 mM to 0.5 mM, to inactivate the CaSR, did not significantly inhibit basal secretion at 2 mM glucose. However, at 10 mM glucose, low [Ca²⁺]_o significantly inhibited secretion (P = 0.002), as would be expected from the decrease in I_Ca. Addition of 5 mM L-His to the low [Ca²⁺]_o solutions had no significant effect at 2 mM glucose but potentiated secretion by 34% at 10 mM glucose (P = 0.02). This partial inhibition of the potentiation of insulin secretion at low [Ca²⁺]_o is consistent with a role for the CaSR but indicates that CaSR sensitization is not the sole mechanism through which L-His exerts its effects. Addition of 10 µM Cd²⁺ to the 2.6 mM [Ca²⁺]_o solutions had no significant effect on secretion at either 2 mM or 10 mM glucose (Fig. 2B) despite producing an equivalent reduction of I_Ca to the 0.5 mM [Ca²⁺]_o solutions. This lack of effect of Cd²⁺ on insulin secretion was unexpected given the decrease in I_Ca and may reflect the ability of the residual I_Ca to support secretion in the presence of maintained CaSR activity.

View larger version (34K): [in this window] [in a new window]   FIG. 2. Reducing extracellular [Ca2+] or addition of Cd2+ both reduce Ca2+ currents but have different effects on insulin secretion. A, Reduction of extracellular [Ca2+] from 2.6 mM to 0.5 mM or addition of 10 µM Cd2+ (to a 2.6 mM [Ca2+] solution) produce a similar, reversible, inhibition of INS-1 cell Ca2+ currents. Current amplitudes averaged from six cells are shown in the left panel and representative current-voltage relations from a cell before, during and after washout of 10 µM Cd2+ is shown in the right panel. B, Insulin secretion from INS-1 cells in normal (2.6 mM) [Ca2+] at 2 mM or 10 mM glucose in the presence or absence of 5 mM L-His is shown in columns 1–4. Columns 5–8 show secretion in 0.5 mM [Ca2+] solutions. Cd2+ (10 µM) did not significantly inhibit secretion at either 2 mM or 10 mM glucose (columns 9–12).

The pathway(s) by which the CaSR signals in ß-cells is not known but is reported not to involve the heterotrimeric G-proteins Gs, Gi, Go, Gq/11, or the activation of PLC (3, 11). We have sequenced the full open reading frame for the cDNA of the CaSR expressed in INS-1 cells, this sequence is identical with that from rat nerve terminals (data not shown; and Ref. 8) and the translated protein sequence is identical with that of the parathyroid cell receptor. The parathyroid CaSR expressed in HEK-293 cells activates PLC signaling and inhibits cAMP production induced by Gs coupled receptors (13). GLP-1 is an important incretin hormone that potentiates glucose-induced insulin secretion and the GLP-1R activates Gs and stimulates cAMP production (23). Although the CaSR does not directly stimulate or inhibit cAMP production in ß-cells (3, 11), we were interested to test whether the CaSR modulates GLP-1R activity. To address this question, we transfected HEK-293 cells with a cAMP reporter, pCRE-Luc. This reporter consists of the luciferase gene driven by a multimerized cAMP response element (CRE). Control experiments were initially performed to test whether HEK-293 cells show a pCRE-Luc response to GLP-1 or to CaSR agonists when transfected with pCRE-Luc alone. No pCRE-Luc response was observed when GLP-1 was applied to these cells at concentrations up to 10^-6 M (data not shown) and no significant increase in luciferase activity was recorded when [Ca²⁺]_o was increased from 1.8 mM to 5 mM or 10 mM (data not shown). These control experiments indicate that the HEK-293 cells have no endogenous pCRE-Luc response to the GLP-1R and CaSR agonists without cotransfection of the relevant receptor. Experiments were then performed in which pCRE-Luc was cotransfected into HEK-293 cells with the GLP-1R, or with the GLP-1R and the CaSR. HEK-293 cells cotransfected with the GLP-1R and pCRE-Luc showed a dose-dependent increase in reporter activity in response to GLP-1 (7–36)amide. Detailed experiments were performed using four concentrations of GLP-1 selected from the full dose-response curve; control (no GLP-1), 10^-11 M (threshold of activation), 10^-9 M (approximate EC₅₀) and 10^-7 M (maximal response). These concentrations were selected to test for effects on maximal cAMP production and to test for shifts of the dose-response curve. Figure 3A shows data from HEK-293 cells cotransfected with the GLP-1R and pCRE-Luc. At 1.8 mM [Ca²⁺]_o, addition of 5 mM L-His had no significant effect on GLP-1 stimulated cAMP-reporter activity. Increasing [Ca²⁺]_o to 5 mM had no significant effect on cAMP-reporter activity in the absence of GLP-1, but maximal GLP-1-stimulated activity was increased by about 40% (P < 0.001). Addition of 5 mM L-His to the 5 mM [Ca²⁺]_o solution had no significant effect on basal pCRE-Luc activity but maximal GLP-1 stimulated cAMP production was increased by about 80% (P < 0.001). Figure 3B shows data from cells transfected with the CaSR in addition to the GLP-1R and pCRE-Luc. L-His had no significant effect on cAMP-reporter activity at 1.8 mM [Ca²⁺]_o, but the increase in reporter activity seen at 5 mM [Ca²⁺]_o with the GLP-1R alone (Fig. 3A) was abolished by cotransfection with the CaSR (Fig. 3B). These data are consistent with the previously reported coupling of the CaSR to Gi/o and inhibition of cAMP production (13). One consequence predicted from this coupling to Gi/o is that amino acids acting on the CaSR should increase the Ca²⁺-induced inhibition of GLP-1-induced cAMP production. However, when 5 mM L-His was added to the 5 mM [Ca²⁺]_o solution, a significant (P = 0.005) increase in reporter activity was observed (Fig. 3B), similar to the effect seen with the GLP-1R alone (Fig. 3A). Similar data were obtained using 5 mM L-Leu (Fig. 4), an amino acid that is ineffective on the CaSR. L-Leu had no significant effect at 1.8 mM [Ca²⁺] in cells transfected with the GLP-1R alone (Fig. 4A) or when cotransfected with the CaSR (Fig. 4B). Increasing [Ca²⁺] to 5 mM significantly (P = 0.005) increased reporter activity in response to 10^-7 M GLP-1 in cells expressing the GLP-1R alone but had no effect in cells cotransfected with the CaSR. At 5 mM [Ca²⁺], addition of L-Leu significantly potentiated GLP-1-induced reporter activity both in cells expressing the GLP-1R alone (P = 0.002) and in cells cotransfected with the CaSR (P = 0.005). These data suggest that amino acid effects on GLP-1R signaling dominate their effects mediated through CaSR coupling to Gi/o.

View larger version (20K): [in this window] [in a new window]   FIG. 3. HEK-293 cells were transfected with the pCRE-Luc cAMP reporter and the GLP-1R (A) or with the GLP-1R and CaSR (B). Cells were incubated in DMEM culture medium with four concentrations of GLP-1 (7–36)amide for 4 h and luciferase activity (relative light units, RLU) measured. GLP-1 produced a dose-dependent increase in reporter activity under all conditions. Horizontal bars indicate [Ca2+] and the presence of absence of L-His and apply to both (A) and (B). All data are averaged from three transfections with a total of 36 samples per point. Statistical probability values are indicated as *, 0.005; **, <0.001.

View larger version (20K): [in this window] [in a new window]   FIG. 4. This figure shows data from HEK-293 cells transfected and treated using the same protocols as in Fig. 3. The activity of the cAMP reporter in response to stimulation with GLP-1 (7–36) amide was measured at 1.8 mM and 5 mM [Ca2+] in the presence of absence of 5 mM L-Leu, an amino acid that has no effect on the CaSR. The data from cells treated with L-Leu are similar to those shown in Fig. 5 with L-His. All data are averaged from three transfections with a total of 36 samples per point. Statistical probability values are indicated as *, 0.005; **, 0.002.

View larger version (25K): [in this window] [in a new window]   FIG. 5. A, Control cAMP reporter activity from HEK-293 cells transfected with the GLP-1R (left panel) or cotransfected with the GLP-1R and CaSR (right panel). Cells were stimulated with GLP-1 (7–36)amide for 4 h and cAMP reporter activity measured. A, Cells transfected with the GLP-1R showed a dose-dependent increase in cAMP production that was increased in 5 mM [Ca2+] compared with 1.8 mM [Ca2+]. Cotransfection with the CaSR inhibited this [Ca2+]-dependent increase in cAMP production. B, Data from cells treated overnight with PTX. Cells transfected with the GLP-1R again showed increased cAMP production at 5 mM [Ca2+]. This potentiation was not inhibited in PTX-treated cells cotransfected with the CaSR, and these cells showed an increase in cAMP at 5 mM [Ca2+]. Each point represents the mean of 12 samples. Statistical probability values are indicated as *, 0.007; **, <0.001.

View larger version (22K): [in this window] [in a new window]   FIG. 6. GLP-1-induced CRE-Luc reporter activity is potentiated by high [Ca2+] and L-His in INS-1 cells. INS-1 cells express both the GLP-1R and CaSR. However, unlike in HEK-293 cells cotransfected with these two receptors, [Ca2+] potentiated GLP-1-induced reporter activity and 5 mM L-His further increased the activity at 5 mM [Ca2+] but not at 1 mM [Ca2+]. The effects of [Ca2+] and L-His were similar at both 2 mM and 10 mM glucose but the basal activity was approximately 50% higher at 10 mM glucose. Data shown is normalized to the activity in 1 mM [Ca2+] with no GLP-1 added and is averaged from six transfections with a total of 42 samples. Statistical probabilities calculated using one-way ANOVA are: P1 = 0.009; P2 = 0.037; P3 < 0.001; P4 = 0.018.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It has been demonstrated that amino acid levels found after feeding induce a left-shift of the glucose dependence of insulin secretion (19). We propose that the physiological role of the CaSR in ß-cells is to function as an amino acid sensor to potentiate glucose-induced secretion after a normal, mixed meal. This is certainly not the only mechanism through which amino acids exert their effects, nor is it likely to be the only receptor-mediated mechanism. The purinergic P2Y1 receptor is amino acid sensitive (17), is expressed in ß-cells, and purinergic agonists potentiate insulin secretion from rat and human islets in a glucose-dependent manner (25, 26, 27), similar to R-467 and GLP-1. Additionally, we show that leucine and histidine can also increase GLP-1-induced cAMP reporter activity. These multiple receptor-mediated effects may contribute to the increased sensitivity of insulin secretion to glucose in the presence of amino acids (19).

The signaling pathways activated by the CaSR in ß-cells have not been described, although negative data has argued against a role for Gs, Gq/11, Gi, and Go (3, 11). These data raised the possibility that the ß-cell may express a unique form of the CaSR, and we therefore sequenced the full open reading frame of the CaSR from INS-1 cell cDNA. We found the cDNA sequence to be identical with that expressed in rat nerve terminals (8) and the predicted amino acid sequence is identical with the receptor from the parathyroid gland. This receptor couples to Gq/11 and Gi/o, stimulates intracellular calcium mobilization and inhibits cAMP production (13). This coupling is consistent with reports that human insulinoma cells respond to elevated extracellular [Ca²⁺] with a thapsigargin-sensitive increase of intracellular [Ca²⁺] (4). The reason why previous studies have failed to identify the activation of Gq/11 in ß-cells is not clear but may be related to the relatively low expression level of the receptor in these cells. We performed quantitative PCR using INS-1 cell cDNA and these data indicate that the abundance of RNA for the CaSR is about 6-fold lower than that for the GLP-1R.

Activation of the CaSR has also been shown to inhibit cAMP production induced by several Gs-coupled hormone receptors, including the glucagon receptor (13, 28). Coexpression of the CaSR and the GLP-1R in HEK-293 cells reveals that elevated extracellular [Ca²⁺] produces a PTX-sensitive inhibition of GLP-1-induced cAMP accumulation, consistent with CaSR coupling to Gi. An unexpected observation during these studies was that L-His did not inhibit cAMP production, as might be expected, but produced an overall potentiation of GLP-1-induced cAMP reporter activity. The potentiation of cAMP reporter activity by L-His and by L-Leu was observed when the GLP-1 receptor was expressed alone. These observations suggest that GLP-1R signaling is sensitive to amino acids and that these nutrients may potentiate GLP-1 and glucose-dependent insulin secretion through a direct action on GLP-1R signaling, in addition to effects on the CaSR and the P2Y1 receptor. The opposite effects on cAMP production produced by two agonists of the CaSR, Ca²⁺ and L-His, may result from a greater effect on GLP-1R- than on the CaSR-signaling. Experiments using INS-1 cells showed a potentiation of GLP-1-induced cAMP reporter activity by both Ca²⁺ and L-His and these results may reflect the higher expression level of the GLP-1R compared with the CaSR in these cells. Raising [Ca²⁺]_o from 1–5 mM potentiated GLP-1-induced cAMP reporter activity in INS-1 cells, whereas increasing [Ca²⁺]_o did not produce such a potentiation in HEK-293 cells coexpressing the GLP-1R and CaSR (Fig. 6). This observation would suggest that CaSR coupling to Gi/o might not have a significant effect on insulin secretion from INS-1 cells. Whether this difference reflects the relative expression levels of the two receptors in INS-1 and transfected HEK-293 cells or a difference in the efficacy of coupling to Gi/o in the two cell lines remains uncertain.

It is interesting that Ca²⁺ and amino acids stimulate different patterns of [Ca²⁺] oscillation when the CaSR is expressed in HEK-293 cells, and it was suggested that this difference in activity might reflect the ability of the CaSR to discriminate between different agonists (29). It is possible, therefore, that an alternative explanation for the different effects of Ca²⁺ and L-His on cAMP production is that they might differentially regulate coupling of the CaSR to Gq/11 and Gi/o. This difference between Ca²⁺ and L-His effects on cAMP production might be important for a physiological role of the CaSR in islets where [Ca²⁺]_o is not known to substantially change but amino acids levels do increase after a normal meal.

The calcium sensitivity of the parathyroid CaSR is increased by a range of amino acids with different efficacies (17). Whether the potentiation of cAMP production by amino acids after activation of the GLP-1R occurs through a direct interaction with the receptor, or through effects on downstream effectors, remains to be determined. However, a direct effect of amino acids on the GLP-1R, in addition to effects on the CaSR and P2Y1 receptors (17), would represent the third type of receptor expressed in ß-cells that potentiates glucose-dependent insulin secretion and is amino acid sensitive.

In summary, amino acid-induced potentiation of both GLP-1-induced cAMP production and activation of PLC by the CaSR will potentiate insulin secretion. Activation of these two signaling pathways results in the augmentation of both ATP-sensitive potassium channel-dependent and -independent glucose-induced secretion (30). We propose that amino acids modulate glucose-dependent insulin secretion, at least in part, through effects on CaSR, GLP-1R, and P2Y1 receptor signaling. The differential sensitivity of these receptors to different amino acids may also contribute to the differential regulation of secretion by different amino acids (31) and the increased sensitivity of insulin secretion to glucose in the presence of amino acids (19).

	Acknowledgments

 
We thank Maurice Castonguay and Karen McManus for expert technical assistance, Dr. Claus Wollheim for providing the INS-1 cells, and Dr. Mei Bai for the CaSR expression vector.

	Footnotes

 
This work was supported in part by United States Public Health Service Grant DK30834 (to J.F.H.). J.F.H. is an investigator with the Howard Hughes Medical Institute.

Abbreviations: BCH, 2-Amino-2-norbornane-carboxylic acid; CaSR, calcium-sensing receptor; CRE, cAMP response element; Ct, threshold cycle; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLP-1, glucagon-like peptide-1; GLP-1R, GLP-1 receptor; HEK, human embryonic kidney; PLC, phospholipase C; PTX, pertussis toxin; QPCR, quantitative PCR; SES, standard extracellular solution.

Received April 21, 2003.

Accepted for publication July 15, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Flatt PR, Berggren PO, Gylfe E, Hellman B 1980 Calcium and pancreatic-cell function. IX. Demonstration of lanthanide-induced inhibition of insulin secretion independent of modifications in transmembrane Ca²⁺ fluxes. Endocrinology 107:1007–1013[Abstract]
2. Wang J, Morley P, Begin-Heick N, Whitfield JF 1995 Do pancreatic islet cells from neonatal rats have surface receptors or sensors for divalent cations? Cell Signal 7:651–658[CrossRef][Medline]
3. Squires PE, Harris TE, Persaud SJ, Curtis SB, Buchan AMJ, Jones PM 2000 The extracellular calcium-sensing receptor on human ß-cells negatively modulates insulin secretion. Diabetes 49:409–417[Abstract]
4. Kato M, Doi R, Imamura M, Furutani M, Hosotani R, Shimada Y 1997 Calcium-evoked insulin release from insulinoma cells is mediated via calcium-sensing receptor. Surgery 122:1203–1211[CrossRef][Medline]
5. Aida K, Koishi S, Tawata M, Onaya T 1995 Molecular cloning of a putative Ca(2+)-sensing receptor cDNA from human kidney. Biochem Biophys Res Commun 214:524–529[CrossRef][Medline]
6. Rasschaert J, Malaisse WJ 1999 Expression of the calcium-sensing receptor in pancreatic islet ß-cells. Biochem Biophys Res Commun 264:615–618[CrossRef][Medline]
7. Bruce JI, Yang X, Ferguson CJ, Elliot AC, Steward MC, Case RM, Riccardi D 1999 Molecular and functional identification of a Ca²⁺ (polyvalent cation)-sensing receptor in rat pancreas. J Biol Chem 274:20561–20568[Abstract/Free Full Text]
8. Ruat M, Molliver ME, Snowman AM, Snyder SH 1995 Calcium sensing receptor: molecular cloning in rat and localization to nerve terminals. Proc Natl Acad Sci USA 92:3161–3165[Abstract/Free Full Text]
9. Riccardi D, Park J, Lee WS, Gamba G, Brown EM, Hebert SC 1995 Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor. Proc Natl Acad Sci USA 92:131–135[Abstract/Free Full Text]
10. Malaisse WJ, Louchami K, Laghmich A, Ladriere L, Morales M, Villaneuva-Penacarrillo ML, Valverde I, Rasschaert J 1999 Possible participation of an islet ß-cell calcium-sensing receptor in insulin release. Endocrine 11:293–300[CrossRef][Medline]
11. Straub SG, Kornreich B, Oswald RE, Nemeth EF, Sharp GW 2000 The calcimimetic R-467 potentiates insulin secretion in pancreatic ß-cells by activation of a non-specific cation channel. J Biol Chem 275:18777–18784[Abstract/Free Full Text]
12. Kato M, Doi R, Imamura M, Okada N, Shimada Y, Hosotani R, Miyazaki JI 1998 Response of human insulinoma cells to extracellular calcium is different from normal B cells. Dig Dis Sci 43:2429–2438[CrossRef][Medline]
13. Brown EM, MacLeod RJ 2001 Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 81:239–297[Abstract/Free Full Text]
14. Imaizumi T, Kocsis JD, Waxman SG 1999 The role of voltage-gated Ca²⁺ channels in anoxic injury of spinal cord white matter. Brain Res 817:84–92[CrossRef][Medline]
15. Wang JP, Needleman DH, Seryshev AB, Aghdasi B, Slavik KJ, Liu SQ, Pedersen SE, Hamilton SL 1996 Interaction between ryanodine and neomycin binding sites on Ca²⁺ release channel from skeletal muscle sarcoplasmic reticulum. J Biol Chem 271:8387–8393[Abstract/Free Full Text]
16. Lei S, Dryden WF, Smith PA 1998 Involvement of Ras/MAP kinase in the regulation of Ca²⁺ channels in adult bullfrog sympathetic neurons by nerve growth factor. J Neurophysiol 80:1352–1361[Abstract/Free Full Text]
17. Conigrave AD, Quinn SJ, Brown EM 2000 L-Amino acid sensing by the extracellular Ca²⁺-sensing receptor. Proc Natl Acad Sci USA 97:4814–4819[Abstract/Free Full Text]
18. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC 1993 Cloning and characterization of an extracellular Ca²⁺-sensing receptor from bovine parathyroid. Nature 366:575–580[CrossRef][Medline]
19. Bolea S, Pertusa JA, Martin F, Sanchez-Andres JV, Soria B 1997 Regulation of pancreatic ß-cell electrical activity and insulin release by physiological amino acid concentrations. Pflugers Arch 433:699–704[CrossRef][Medline]
20. Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-C(T)) method. Methods 25:402–408[CrossRef][Medline]
21. Gao ZY, Li G, Najafi H, Wolf BA, Matschinsky FM 1999 Glucose regulation of glutaminolysis and its role in insulin secretion. Diabetes 48:1535–1542[Abstract]
22. Ashcroft FM, Rorsman P 1989 Electrophysiology of the pancreatic ß-cell. Prog Biophys Mol Biol 54:87–143[CrossRef][Medline]
23. Kieffer TJ, Habener JF 1999 The glucagon-like peptides. Endocr Rev 20:876–913[Abstract/Free Full Text]
24. Firsov D, Aarab L, Mandon B, Siaume-Perez S, de Rouffignac C, Chabardes D 1995 Arachidonic acid inhibits hormone-stimulated cAMP accumulation in the medullary thick ascending limb of the rat kidney by a mechanism sensitive to pertussis toxin. Pflugers Arch 429:636–646[CrossRef][Medline]
25. Fernandez-Alvarez J, Hillaire-Buys D, Loubatieres-Mariani MM, Gomis R, Petit P 2001 P2 receptor agonists stimulate insulin release from human pancreatic islets. Pancreas 22:69–71[CrossRef][Medline]
26. Hillaire-Buys D, Chapal J, Bertrand G, Petit P, Loubatieres-Mariani MM 1994 Purinergic receptors on insulin-secreting cells. Fundam Clin Pharmacol 8:117–127[Medline]
27. Petit P, Hillaire-Buys D, Manteghetti M, Debrus S, Chapal J, Loubatieres-Mariani MM 1998 Evidence for two different types of P2 receptors stimulating insulin secretion from pancreatic B cell. Br J Pharmacol 125:1368–1374[CrossRef][Medline]
28. Bapty BW, Dai LJ, Ritchie G, Canaff L, Hendy GN, Quamme GA 1998 Mg²⁺/Ca²⁺ sensing inhibits hormone-stimulated Mg²⁺ uptake in mouse distal convoluted tubule cells. Am J Physiol 275:F353–F360
29. Young SH, Rozengurt E 2002 Amino acids and Ca²⁺ stimulate different patterns of Ca²⁺ oscillations through the Ca²⁺-sensing receptor. Am J Physiol Cell Physiol 282:C1414–22
30. Straub SG, Sharp GW 2002 Glucose-stimulated signaling pathways in biphasic insulin secretion. Diabetes Metab Res Rev 18:451–463[CrossRef][Medline]
31. Strandgaard C, Curry DL 1998 Differential insulin secretory responses to cationic and branched-chain amino acids. Pancreas 17:65–71[Medline]

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