MuscarinicControlofMIN6Pancreatic …Signaling by the M3R also activates ERK1/2 in cells, most likely downstream of elevated intracellular calcium (12–14). ERK1/2 activation enhances

Muscarinic Control of MIN6 Pancreatic � Cells Is Enhanced byImpaired Amino Acid Signaling*

Received for publication, March 13, 2014 Published, JBC Papers in Press, April 2, 2014, DOI 10.1074/jbc.M114.565069

Marcy L. Guerra1, Eric M. Wauson, Kathleen McGlynn, and Melanie H. Cobb2

From the Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9041

Background: Depletion of the GPCR T1R1/T1R3 increased calcium and ERK1/2 signaling by carbachol.Results: T1R3 depletion or reducing amino acids overnight increased M3 muscarinic receptor expression and altered calciumresponses.Conclusion: M3 receptor expression in � cells is up-regulated by reduced amino acid availability.Significance: The M3 muscarinic receptor is a potential therapeutic target in � cells with impaired amino acid sensitivity.

We have shown recently that the class C G protein-coupledreceptor T1R1/T1R3 taste receptor complex is an early aminoacid sensor in MIN6 pancreatic � cells. Amino acids are unableto activate ERK1/2 in � cells in which T1R3 has been depleted.The muscarinic receptor agonist carbachol activated ERK1/2better in T1R3-depleted cells than in control cells. Ligands thatactivate certain G protein-coupled receptors in pancreatic �cells potentiate glucose-stimulated insulin secretion. Amongthese is the M3 muscarinic acetylcholine receptor, the majormuscarinic receptor in � cells. We found that expression of M3receptors increased in T1R3-depleted MIN6 cells and that cal-cium responses were altered. To determine whether thesechanges were related to impaired amino acid signaling, we com-pared responses in cells exposed to reduced amino acid concen-trations. M3 receptor expression was increased, and some, butnot all, changes in calcium signaling were mimicked. These find-ings suggest that M3 acetylcholine receptors are increased in �cells as a mechanism to compensate for amino acid deficiency.

The essential function of the pancreatic � cell is to secreteinsulin in response to increases in circulating glucose. Othernutrients, hormones, and paracrine agents influence pancre-atic � cell functions and insulin secretion to optimize glu-cose homeostasis. Ligands for several G protein-coupled recep-tors (GPCRs)3 are among the most significant in tuninginsulin secretion from � cells, which express several differ-ent classes of GPCRs, including muscarinic acetylcholinereceptors (mAChRs) (1).

Five mAChRs subtypes, M1-M5, have been identified (2).The M1, M3, and M5 subtypes are Gq-coupled receptors,whereas M2 and M4 are Gi-coupled receptors that are inhibitedby pertussis toxin (3). � cell muscarinic receptors are Gq-cou-pled because binding of acetylcholine to these receptors resultsin the well characterized action of Gq to activate phospholipaseC �. The resulting hydrolysis of phosphatidylinositol 4,5-bis-phosphate generates the second messenger inositol 1,4,5-triphosphate, which binds to its receptor on the ER and inducescalcium release from intracellular stores (4). Muscarinic ago-nist-induced mobilization of intracellular calcium (Ca2�

i) wasabsent in mice selectively lacking two members of the � cell Gqprotein family, G�q and G�11 (5).

The M3 mAChR (M3R) is the predominant receptor subtypeexpressed in � cells and insulin-secreting cell lines (6, 7). Para-sympathetic nerve endings that innervate the pancreas releaseacetylcholine during the preabsorptive and absorptive phasesof feeding (8) to activate this receptor. Studies utilizing M3Rknockout mice have implicated M3R as the receptor subtyperesponsible for cholinergic potentiation of glucose-stimulatedinsulin secretion (9, 10). Furthermore, mice selectively deficientin � cell M3Rs demonstrated impaired glucose tolerance andreduced insulin release, whereas mice overexpressing M3Rs in� cells exhibited a significant increase in glucose tolerance andinsulin release (11). Similar observations were made in miceoverexpressing constitutively active � cell M3Rs (1).

Signaling by the M3R also activates ERK1/2 in � cells, mostlikely downstream of elevated intracellular calcium (12–14).ERK1/2 activation enhances insulin gene transcription follow-ing nutrient-induced insulin secretion (15–18). We havereported previously that the GPCR complex T1R1/T1R3 is anearly amino acid sensor in the MIN6 pancreatic � cell line andin other cell types (12). Similar to M3R, T1R1/T1R3 activationleads to a rise in Ca2�

i and ERK1/2 phosphorylation that ispartially dependent upon phospholipase C � activation.Reduced expression of T1R3 in MIN6 cells resulted in adecrease of amino acid-induced ERK1/2 and mammalian targetof rapamycin complex 1 activation. Signaling defects in cells inwhich the receptor had been depleted included a reduction inthe ability of amino acids to induce changes in Ca2�

i (12).Despite the impaired ability of amino acids to stimulate

ERK1/2 in T1R3-depleted MIN6 cells, carbachol, a muscarinic

* This work was supported, in whole or in part, by National Institutes of HealthGrant DK55310. This work was also supported by Robert A. Welch Founda-tion Grant I1243.

1 Supported by a mentor-based postdoctoral fellowship from the AmericanDiabetes Association during the initial part of this study.

2 To whom correspondence should be addressed: Dept. of Pharmacolo-gy, UT Southwestern Medical Center, 6001 Forest Park Rd., Dallas, TX75390-9041. Tel.: 214-645-6122; Fax: 214-645-6124; E-mail: [email protected].

3 The abbreviations used are: GPCR, G protein-coupled receptor; mAChR,muscarinic acetylcholine receptor; ER, endoplasmic reticulum; M3R, M3muscarinic acetylcholine receptor; 2-APB, 2-aminoethoxydiphenyl borate;SOCE, store-operated calcium entry; ROCE, receptor-operated calciumentry; pERK, phospho-ERK; RGS, regulator of G protein signaling; SOCC,store-operated calcium channel; KRBH, Krebs-Ringer bicarbonate solution.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 20, pp. 14370 –14379, May 16, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

14370 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 20 • MAY 16, 2014

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receptor agonist, activated ERK1/2 better in T1R3-depletedcells than in control cells (12). We explored the underlyingmechanisms for the enhanced carbachol response in MIN6cells to determine whether similar mechanisms were enlisted tocompensate for amino acid deficiency.

EXPERIMENTAL PROCEDURES

Materials—Fura-2/AM was purchased from MolecularProbes. Nifedipine was purchased from Calbiochem. 2-Amino-ethoxydiphenyl borate (2-APB) was purchased from Sigma.Thapsigargin was purchased from Santa Cruz Biotechnology.

Cell Culture—MIN6 cells were cultured, and stable cell lineswith T1R3 expression reduced following expression of a shorthairpin were created and maintained as described previously(12).

Calcium Assays—Cells were plated at 80% confluency inwhite-walled, 96-well plates (Costar 3903). After 48 h, the cellswere washed twice with PBS (0.2 ml/well) and incubated with 5�M Fura-2/AM diluted in Krebs-Ringer bicarbonate solution(KRBH) containing 115 mM NaCl, 5 mM KCl, 24 mM NaHCO3,1 mM MgCl2, 2.5 mM CaCl2, 25 mM HEPES (pH 7.4), 0.1% BSA,and 4.5 mM glucose for 1 h (0.1 ml/well). Cells were then washedtwice with KRBH (0.2 ml/well) and equilibrated in the samebuffer for 30 min (0.1 ml/well). Agents were applied (0.1ml/well) to triplicate wells at 2� concentrations using injectorsat a rate of 225 �l/s. Changes in Ca2�

i were assessed every 0.74 sby dual excitation of Fura-2 at 340/11 and 380/20 nm (center/bandpass) and emission at 508/20 nm using the SynergyTM 2multimode microplate reader (BioTek) with Gen5TM software.Cells were pretreated with the indicated inhibitors for 30 minprior to stimulation. For experiments performed in the absenceof calcium, cells were loaded, washed, and equilibrated withcalcium-free KRBH in which MgCl2 was substituted for 2.5 mM

CaCl2. To assess store-operated calcium entry (SOCE), intra-cellular stores were depleted using 10 �M thapsigargin. Calciumwas then replenished with a second injection of KRBH contain-ing 12.5 mM CaCl2 (5� concentration, 50 �l/well). To assessreceptor-operated calcium entry (ROCE), after calcium reple-tion, a third injection was required to apply 0.6 mM carbachol(6� concentration, 50 �l/well). Final concentrations of allagents were 1�. For experiments involving nifedipine or2-APB, cells were pretreated with inhibitors for 30 min prior tostimulation. All steps in each assay were performed at roomtemperature.

Nutrient Deprivation—MIN6 cells were plated as above forcalcium assays or in 12-well plates for RNA or protein isolation.To examine the effects of reduced amino acids, cells nearingconfluency were washed twice with PBS and incubated withKRBH supplemented with 10% dialyzed serum, 4.5 mM glucose,and either 1.0� amino acids (12) or 0.1� amino acids for 16 h at37 °C and 10% CO2 prior to stimulation with carbachol or celllysis. Calcium was measured as above with reduced amino acidsthroughout. To examine the effects of reduced glucose, cellswere incubated as above in KRBH containing 10% dialyzedserum, 1� amino acids, and either 25 or 2 mM glucose. Humanislets were provided by the Integrated Islet Distribution Pro-gram. Islets were washed twice in KRBH and then once in

KRBH containing 10% dialyzed serum, 4.5 mM glucose, andeither 0.1� or 1� amino acids prior to treatment overnight.

Immunoblotting—Cells were lysed in 50 mM HEPES (pH 7.5),150 mM NaCl, 1% Triton X-100, 10 �g/ml aprotinin, 5 �g/mlleupeptin, 5 �g/ml pepstatin A, 0.2 mg/ml PMSF, 100 mM NaF,and 2 mM Na3VO4. For lysates from stable cell lines, 40 �g ofprotein, as determined by BCA assay (Pierce), was resolved bypolyacrylamide gel electrophoresis in sodium dodecyl sulfateand transferred to nitrocellulose membranes. Membranes wereblocked with 5% nonfat milk in Tris-buffered saline containing0.1% Tween 20 (TBST) for 2 h at room temperature. Mem-branes were incubated with primary antibodies overnight at4 °C. Antibodies were diluted in 5% milk/TBST as follows: M3Rmuscarinic receptor (1:250, Millipore, catalog no. AB9018, rab-bit, polyclonal) and ERK1/2 (1:2000, Abcam, catalog no.ab54230, mouse, monoclonal). For the carbachol time course,pERK1/2/ERK1/2 blots used 20 �g of protein. Antibodies werediluted in 5% milk/TBST as follows: pERK1/2 (1:1000, Sigma,catalog no. M8159, mouse, monoclonal) and ERK1/2 (1:1000,691 rabbit (19)). The membranes were then washed with TBSTand incubated for 1 h at room temperature with secondary anti-bodies: donkey anti-rabbit IRDye 680RD or donkey anti-mouseIRDye 800CW (1:10,000, Li-Cor Biosciences). The membraneswere washed with TBST and then imaged using the LI-CorOdyssey infrared imaging system. Blots were quantified usingLi-Cor Odyssey application software (version 3.0).

RNA Isolation, cDNA Synthesis, and Real-time QuantitativePCR—Cells or human islets were harvested in TRI reagent�solution, and RNA was extracted according to the instructionsof the manufacturer (Applied Biosystems). cDNA was gener-ated using a high-capacity cDNA reverse transcription kit(Applied Biosystems). SYBR Green Supermix with ROX waspurchased from Bio-Rad. GAPDH was used as an internalexpression control. The primers were as follows: GAPDH, 5�-CTGGAGAAACCTGCCAAGTA-3� (forward) and 5�-TGTT-GCTGTAGCC GTATTCA-3� (reverse); actin, 5�-AGGTCAT-CACTATTGCAACGA-3� (forward) and 5�-CACTTCATG-ATGGAATTGAATGTAGTT-3� (reverse); M3R (human)muscarinic receptor, 5�-ATTAAGCACTTGTGTTCTGATT-AGT-3� (forward) and 5�-CACGCCACAGCAAAACCTTA-3�(reverse); M1R muscarinic receptor, 5�-CCCTGGCAGGTGG-CCTTC ATC-3� (forward) and 5� AGCACAGGCCAGGCTC-AGCAG-3� (reverse); mouse M3R muscarinic receptor, 5�-ACAGCCACCTGGAG CACGGC-3� (forward) and 5�-AAC-GCAGCACTTCAAGAGGAGAGTC-3� (reverse); M5R mus-carinic receptor, 5�-GGCCAAGAAGAGGGGAGGCCA-3�(forward) and 5�-CCGGGGTGCCGTTGACAGTG-3� (re-verse); G�q, 5�-GCCGACCCTTCCTATCTGC-3� (forward)and 5�-CCCCCTACATCGAC CATTCTGA-3� (reverse);RGS4, 5�-TCTGCCGGCTTCCTGCCTGA-3� (forward) and5�-TCTTGGCTTACCCTCTGGCAAGTT-3� (reverse); andTRPC6, 5�-GGAGACGACGGCTACCCG CA-3� (forward)and 5�-AATCGTCTGCCGCCGGTGAG-3� (reverse). Valid-ated T1R3 primers were purchased from Bio-Rad (unique assayno. qmmuCED0004159).

Statistical Analysis—Results were expressed as means � S.E.determined from three independent experiments. Statisticalsignificance was calculated using Student’s t test.

T1R3 and Muscarinic Signaling

MAY 16, 2014 • VOLUME 289 • NUMBER 20 JOURNAL OF BIOLOGICAL CHEMISTRY 14371


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RESULTS

Carbachol-induced Changes in Ca2�i and M3R Expression

Are Enhanced in MIN6 Cells after Depletion of T1R3—Todetermine the basis for the increased carbachol-inducedERK1/2 phosphorylation in MIN6 cells with reduced T1R3expression (12), we first examined the effect of carbachol onCa2�

i after loading cells with the ratiometric calcium indicatorFura-2. Stimulation of � cells with carbachol produced a bi-phasic rise in Ca2�

i, composed of a rapid and transient peakfollowed by a sustained plateau phase (20, 21). Compared withthe control, we observed a larger rise in peak Ca2�

i in MIN6cells with depleted T1R3 as well as a faster decline during thesecond phase of the carbachol response (Fig. 1, A and B).

One possible reason for enhanced carbachol-inducedchanges in Ca2�

i in MIN6 T1R3 knockdown cells could beincreased expression of mAChRs. Therefore, we performedreal -time quantitative PCR to compare changes in expressionof the Gq-coupled mAChRs, M1, M3, and M5, in MIN6 controland T1R3 knockdown cells. There was 2.35 � 0.06-foldincrease in M3R mRNA in the T1R3knockdown cells compared

with the control, whereas the expression of M1 and M5 recep-tors did not change significantly (Fig. 1C). The increase in M3RmRNA in T1R3 knockdown cells was mirrored by an increase inM3R protein expression that was found by immunoblottinglysates from control and T1R3 knockdown cells (Fig. 1D).Quantitation of the immunoblot analyses indicated a 3.8 � 0.9-fold increase in M3R protein in MIN6 cells with suppressedT1R3 expression (Fig. 1D).

In addition to mAChRs, we also examined the expression ofthe regulator of G protein signaling 4 (RGS4) and transientreceptor potential channel 6 (TRPC6). RGS proteins areGTPase-activating proteins that enhance G�-GTP hydrolysis,thereby decreasing the lifetime of active states of G proteinsubunits, and RGS4 is expressed in � cells (22). TRPC6 has beenreported to be a receptor-operated cation channel that is acti-vated upon GPCR stimulation and subsequent phospholipase C� activation (23, 24). Calcium entry occurring through plasmamembrane channels as a result of GPCR activation indepen-dent of the state of Ca2�

i stores is referred to as ROCE (25). Ithas been demonstrated that carbachol is capable of inducing

FIGURE 1. Carbachol-induced changes in Ca2�i and M3R expression are enhanced in MIN6 cells with reduced T1R3. A, MIN6 control or T1R3 knockdown

cells were loaded with 5 �M Fura-2/AM for 1 h in KRBH containing 4.5 mM glucose and 0.1% BSA. After a 30-min equilibration without Fura-2/AM, cells werestimulated with 100 �M carbachol. 340/380 values were recorded every 0.74 s for 2 min using a microplate reader. Basal 340/380 prestimulation values wereaveraged and subtracted from poststimulation values for each condition. Normalized basal values for treatment with KRBH alone were then subtracted fromthe values obtained with carbachol stimulation to reflect changes in the 340/380 values (expressed as � 340/380). Data are mean � S.E. from three indepen-dent experiments, each in triplicate. B, bar graph representing the mean peak and 2 min 340/380 values � S.E. from data in A. *, p � 0.005; **, p � 0.043; T1R3knockdown compared with control; paired Student’s t test. C, expression of M1, M3, M5, RGS4, and TRPC6 mRNA was measured by quantitative real-time PCRin MIN6 control or T1R3 knockdown cells. Means � S.E. of three independent experiments are shown. *, p � 0.001, cells, paired Student’s t test. D, expressionof M3R protein was detected in three separate sets of lysates (1–3, 50 �g of protein) from MIN6 control or T1R3 knockdown cells by Western blotting (IB). ERK1/2were blotted in the same lysates as the loading control. Blots were quantified using Li-Cor Odyssey application software. Means � S.E. from three independentexperiments are shown. *, p � 0.02, paired Student’s t test. RFU, relative fluorescent units.




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ROCE by activating and promoting cell surface expression ofTRPC6 downstream of muscarinic receptor binding (26, 27).Expression of neither RGS4 nor TRPC6 was significantly differ-ent in T1R3 knockdown cells compared with the control, sug-gesting that the enhanced carbachol response was not a resultof decreased RGS4 or increased TRPC6 expression (Fig. 1C).

Carbachol-induced Changes in Ca2�i in MIN6 Cells with

Suppressed T1R3 Expression Are Largely Dependent on Releaseof Ca2� from Intracellular Stores—The initial rapid rise in cal-cium observed upon carbachol stimulation in � cells has beenshown to be due to the inositol 1,4,5-triphosphate-mediatedrelease of calcium from ER stores, whereas the second phase ismaintained by SOCE (21). SOCE refers to calcium influx thatoccurs through store-operated calcium channels (SOCCs) as aresult of Ca2�

i store depletion (28). We investigated whetherthe contributions of Ca2�

i stores or SOCE to carbachol-in-duced changes in Ca2�

i differed in MIN6 cells with suppressedT1R3 expression compared with control cells. Cells were stim-ulated with carbachol in the absence of extracellular calcium todetermine whether activation of M3R in the T1R3 knockdowncells induced a larger rise in Ca2�

i as a result of the release ofcalcium from intracellular stores. We found that carbacholstimulated a larger rise in Ca2�

i in the absence of extracellularcalcium in the T1R3 knockdown cells compared with controlcells (Fig. 2, A and B). This finding suggests that, in addition toelevated M3R, a larger release of calcium from intracellular

stores may contribute to the enhanced first phase of the carba-chol response in the MIN6 T1R3 knockdown cells.

We next determined whether the larger carbachol-inducedrelease from intracellular stores was a consequence of morecalcium stored in the ER of cells with reduced T1R3 by treatingcells with thapsigargin, an inhibitor of the sarco/endoplasmicreticulum Ca2� ATPase, to deplete ER stores of calcium (29). Inthe absence of extracellular calcium, thapsigargin induced asimilar rise in Ca2�

i in T1R3 knockdown and control cells (Fig.2, C and D). When extracellular calcium was replenished,allowing SOCE to occur, calcium entry through SOCCs wassimilar in both T1R3 knockdown and control cells. We alsoexplored ROCE, after calcium restoration and SOCE hadoccurred, by stimulating cells with carbachol. Despite little orno change in TRPC6 expression in MIN6 cells depleted ofT1R3, ROCE was absent (Fig. 2, C and D), suggesting thatanother, as yet unidentified channel may contribute to ROCE inMIN6 cells.

Consistent with the larger release of calcium from intracel-lular stores induced by carbachol, a greater portion of the peakcarbachol response was sensitive to inhibition by thapsigarginin the T1R3 knockdown cells (Fig. 3A). Thapsigargin blocked agreater portion of carbachol-induced changes in Ca2�

i at 2 minin the control cell line. It is possible that SOCE triggered bycarbachol generates a smaller influx of Ca2� through SOCCs inthe T1R3 knockdown cells compared with control cells. This

FIGURE 2. Carbachol induces a larger release of Ca2� from intracellular stores in MIN6 cells with reduced T1R3 expression. Control or T1R3 knockdowncells loaded with Fura-2/AM. A, cells were stimulated with 100 �M carbachol in calcium-free KRBH. Normalized mean 340/380 values � S.E. from threeindependent experiments, each in triplicate, are shown. B, bar graph of mean peak 340/380 values � S.E. from data in A. *, p � 0.001, paired Student’s t test. C,cells were stimulated with 10 �M thapsigargin in calcium-free KRBH to deplete ER calcium stores. Calcium was replenished by adding a final concentration of2.5 mM CaCl2, allowing SOCE to occur. ROCE was observed by stimulating sequentially with 100 �M carbachol. Normalized mean 340/380 values � S.E. fromthree independent experiments are shown. D, mean peak 340/380 values � S.E. are shown from data in C. *, p � 0.007, paired Student’s t test. RFU, relativefluorescent units.




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might explain why the second phase of the calcium responsedeclined at a faster rate and was less affected by thapsigargin inT1R3 knockdown cells. 2-APB, an inositol 1,4,5-triphosphatereceptor antagonist, inhibited peak and 2 mincarbachol-in-duced rises in Ca2�

i to a similar degree in both control andT1R3 knockdown cells (Fig. 3, A and B). It is conceivable thatthe inhibitory effect of 2-APB on carbachol-stimulated changesin Ca2�

i was due to its actions on SOCCs rather than throughinhibition of inositol 1,4,5-triphosphate receptors (30). Usingan ER-localized FRET sensor, it has been reported that, inMIN6 cells, carbachol-induced reductions in ER calcium werenot inhibited by pretreatment with 2-APB despite lower overallCa2�

i (14). To determine whether or not 2-APB had an effecton SOCE, calcium was depleted and then added back to MIN6cells, as shown in Fig. 2C, and pretreated with 2-APB for 30 min.In 2-APB-treated cells, the ability of thapsigargin to depletestores and the subsequent SOCE was reduced significantly,whereas ROCE was not altered (Fig. 3, C and D).

It has been shown previously that nifedipine, an L-type volt-age-gated calcium channel blocker, inhibits carbachol-inducedchanges in Ca2�

i in MIN6 cells, suggesting that carbacholcauses membrane depolarization (12). It has been proposedthat phosphatidylinositol 4,5-bisphosphate hydrolysis, result-ing from carbachol-induced phospholipase C activation, leadsto decreased KATP channel activity. Decreased KATP channel

activity results in membrane depolarization and activation ofvoltage-gated calcium channels. Nifedipine had a greater effecton the carbachol response in the control cells compared withthose with reduced T1R3, consistent with the observation thatcalcium released from intracellular stores contributes a greaterportion of the carbachol-induced rise in Ca2�

i in the T1R3knockdown cells than does extracellular calcium (Fig. 3, A andB). To determine whether nifedipine was having an effect onSOCE or ROCE, MIN6 cells were pretreated with nifedipine for30 min prior to stimulation. Unlike what was observed with2-APB, nifedipine only had an effect on ROCE (Fig. 3, C and D).This is consistent with the data in Fig. 3, A and B, which dem-onstrated that nifedipine had a smaller effect on carbachol-induced changes in Ca2�

i in the T1R3 knockdown cells inwhich ROCE was absent.

Carbachol-induced Changes in Ca2�i and ERK1/2 Phosphor-

ylation Are Enhanced in MIN6 Cells Deprived of AminoAcids—Because we observed these changes in carbachol sig-naling in MIN6 cells in which T1R3 expression was stablysuppressed and because we have shown previously that theT1R1/T1R3 complex is an early sensor of amino acids, wehypothesized that depriving cells of amino acids may mimicsome of the altered carbachol signaling observed in T1R3knockdown cells. Therefore, we performed experiments inwhich MIN6 cells were incubated in KRBH with 10% dia-

FIGURE 3. Carbachol-induced changes in Ca2�i are largely dependent on release of Ca2� from intracellular stores in MIN6 cells with reduced T1R3

expression. A, MIN6 control or T1R3 knockdown cells loaded with Fura-2/AM were stimulated with 100 �M carbachol after exposure to 10 �M thapsigargin, 50�M 2-APB, or 10 �M nifedipine for 30 min. Normalized mean peak 340/380 values � S.E. from three independent experiments, each in triplicate, are shown. Dataare presented as a percentage of block of the total carbachol response. *, p � 0.001; **, p � 0.02; paired Student’s t test. B, bar graph of the percentage of blockof the total carbachol response at 2 min. ***, p � 0.012; paired Student’s t test. C, wild-type MIN6 cells were pretreated with 50 �M 2-APB or 10 �M nifedipinefor 30 min and then stimulated as shown in Fig. 2C. Normalized mean 340/380 values � S.E. from three independent experiments are shown. D, mean peak340/380 values � S.E. are shown for data in C. *, p � 0.007; **, p � 0.002; ***, p � 0.001; inhibitor compared with the control; paired Student’s t test. RFU, relativefluorescent units.




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lyzed serum, 4.5 mM glucose, and either 1.0� amino acids,representing the full complement of amino acids found inDulbecco’s modified Eagle’s medium, or 0.1� amino acidsfor 16 h to approximate amino acid starvation. Cells werethen stimulated with carbachol for 1, 2, or 5 min, andpERK1/2 were assessed by immunoblotting. We observedlower basal pERK1/2 following amino acid deprivation com-pared with that in cells in complete medium (Fig. 4A). ThepERK1/2 in carbachol-stimulated cells was approximatelythe same in both cell lines. However, the low basal activityresulted in a larger fold increase in pERK1/2 induced by car-bachol in the amino acid-deprived cells compared with con-trol cells (Fig. 4B).

We next determined whether the enhanced effect of carba-chol on ERK1/2 phosphorylation was paralleled by a larger risein Ca2�

i in amino acid-deprived cells. Indeed, a larger rise inpeak Ca2�

i was observed with carbachol stimulation in MIN6cells deprived of amino acids compared with non-deprived cells(Fig. 4, C and D). Although similar, the change was not as greatas that observed in MIN6 T1R3 knockdown cells (Fig. 1, A andB). We also noted significantly lower basal calcium in thedeprived cells compared with non-deprived cells (average basal340/380 value, 0.1� amino acids � 0.2200 � 1.191e-4; 1.0�amino acids � 0.2321 � 1.493e-4; p � 0.001), which may offeran explanation for the lower basal pERK1/2 observed underthese same conditions (Fig. 4A). In addition, although peakCa2�

i induced by carbachol was significantly higher in deprivedcells, the second phase of the response assessed at 2 min did notdiffer (Fig. 4, C and D).

Because M3 receptor expression increased in T1R3 knock-down cells, we wanted to determine whether amino acid dep-rivation also affected M3R expression. As shown in Fig. 5, A andB, M3R mRNA and protein were increased in MIN6 cellsdeprived of amino acids compared with non-deprived cells(1.89 � 0.19- and 1.51 � 0.13-fold, respectively). To determinewhether this was a general response to nutrient deficiency, weexamined effects of lowering the glucose concentration from 25to 2 mM on M3R expression. In this case, we found that M3Rexpression was reduced, not increased (Fig. 5C), indicating adifferential responsiveness of M3R expression to amino acidsand glucose.

We verified, in human islets, that amino acid deprivationaffected M3R expression. As shown in Fig. 5D, M3R expressionwas enhanced 1.5-fold in islets of higher purity (90 and 80%). Inislets of lower purity (70%), no change was observed. The foldincrease of M3R expression in deprived islets was less than thatin deprived MIN6 cells (Fig. 5A, 1.9-fold). This differencemay be due to varied responses among other cell types in islets,in contrast to the relative homogeneity of the MIN6 cell line.

Carbachol-stimulated Mobilization of Calcium from Intra-cellular Stores Is Unaltered in MIN6 Cells Deprived of AminoAcids—We further investigated whether reduced amino acidscould mimic T1R3 knockdown in MIN6 cells by determiningthe ability of carbachol to induce the release of calcium fromintracellular stores under these conditions. In contrast to thesignificantly enhanced ability of carbachol to stimulate therelease of calcium from stores in T1R3 knockdown cells (Fig. 2,A and B), there was no difference in the release from stores

FIGURE 4. Carbachol-stimulated ERK1/2 phosphorylation and changes in Ca2�i are enhanced in MIN6 cells in reduced amino acids. A, MIN6 cells were

in either 1.0� (control) or 0.1� (reduced) amino acids for 16 h. Cells were then stimulated with 100 �M carbachol for the indicated times. Lysates (20 �g ofprotein) were analyzed by Western blotting (IB) to assess pERK1/2 and total ERK1/2. Blots are representative of three independent experiments. B, blots werequantified using Li-Cor Odyssey application software. Data are mean pERK1/2/ERK1/2 ratio � S.E. from three independent experiments. *, p � 0.016; **, p �0.002; paired Student’s t test. C, MIN6 cells as in A were loaded with Fura-2/AM and stimulated with 100 �M carbachol. Data are normalized mean 340/380values � S.E. from three independent experiments, each in triplicate. D, bar graph of mean peak and 2-min 340/380 values � S.E. from data in C. *, p � 0.018;paired Student’s t test. RFU, relative fluorescent units.




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comparing cells with normal and low amino acids (Fig. 6, A andB).

We also investigated whether depletion of intracellular cal-cium stores changed SOCE or ROCE in MIN6 cells deprived ofamino acids to compare with changes in MIN6 T1R3 knock-down and control cells (Fig. 2, C and D). Contrary to what wasobserved in MIN6 cells with depleted T1R3, Ca2�

i resultingfrom store depletion, SOCE, or ROCE was higher in aminoacid-deprived cells compared with non-deprived cells (Fig. 6, Cand D). It is possible that, in amino acid-deprived cells, largeramounts of calcium are stored in the ER because of reducedactivation of T1R3 in the absence of amino acids. The largerrelease of calcium from stores induced by thapsigargin couldcause a greater influx of calcium through SOCCs, consistentwith enhanced SOCE in deprived cells (Fig. 6, C and D). Thealtered ROCE observed in deprived cells could not be associ-ated with an increase in TRPC6 mRNA, again suggesting theinvolvement of another channel in ROCE in MIN6cells (Figs. 5and 6, C and D). Together, these findings indicate that,although M3R expression is up-regulated by lowering aminoacids in the medium, leading to a larger rise in carbachol-in-duced changes in Ca2�

i and ERK1/2 phosphorylation, theeffects on intracellular stores, SOCE, and ROCE that wereobserved in MIN6 T1R3 knockdown cells were not mimicked.Finally, because we showed that fasting increased the expres-sion of T1R3 in mouse tissues (12), we wondered whether

amino acid deficiency also had an effect on T1R3 under theseconditions. We found that expression of T1R3 mRNA wasincreased by incubation in 0.1� amino acids (Fig. 6E).

DISCUSSION

We explored the underlying mechanisms for the enhancedcarbachol response in MIN6 cells following depletion of T1R3and found that there was a significant increase in M3R expres-sion in T1R3-depleted cells. A similar increase in M3R was alsoobserved in amino acid-deprived cells. Thus, the increase inM3R is, at least in part, a response to amino acid deficiency.This change in M3R expression is not observed by reducingglucose. Given the remarkable sensitivity of � cells to changesin glucose concentration, perhaps it is not so surprising thatlowering glucose from 25 to 2 mM, a concentration often used asthe control condition in experiments with these cells, caused adecrease in M3R expression. This result further emphasizesthat the details of how isolated � cells or islets are handled exvivo may have a large impact on signaling capability.

We evaluated the potential impact of other molecules knownto impact � cell function through connections to GPCRs. RGS4terminates signaling from M3Rs, thereby inhibiting their func-tion in MIN6 cells and primary mouse islets (7). It seemed pos-sible that RGS4 might have been suppressed in T1R3 knock-down cells to increase M3R function, but experimental findingsdid not support this idea. We also examined TRPC6 because a

FIGURE 5. Amino acid deprivation increases M3R expression in MIN6 cells. A, expression of M3R and GAPDH mRNAs were assessed by quantitative real-timePCR from amino acid-deprived and control MIN6 cells. Results are mean � S.E. of three independent experiments. *, p � 0.009; paired Student’s t test. B,Western blots (IB) of M3R from 50 �g of lysate protein from amino acid-deprived and control MIN6 cells. ERK1/2 were blotted in the same lysates as the loadingcontrol. Blots were quantified using Li-Cor Odyssey application software. Data are mean M3R/ERK1/2 ratio � S.E. from three independent experiments. p �0.001; paired Student’s t test. C, MIN6 cells were incubated as in Fig. 4A except with either 2 or 25 mM glucose for 16 h. Expression of M3R and GAPDH mRNA wasassessed as in A. Data are mean � S.E. of three independent experiments. *, p � 0.009; paired Student’s t test. D, expression of M3R and actin mRNAs wereassessed as in A from amino acid-deprived and control human islets. The bar graph shows data from three independent sets of human islets of varying purity.




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microarray analysis suggested that it was up-regulated in T1R3knockdown cells. Carbachol is capable of inducing ROCE byactivating and promoting cell surface expression of TRPC6downstream of muscarinic receptor binding (26, 27). Althoughthe suspected change in its mRNA could not be validated,TRPC6 seemed a logical candidate and may be involved in somemanner we did not detect (23, 24).

Differences in M3R signaling between shRNA depletion ofT1R3 and amino acid limitation included effects on the kineticsof changes in intracellular free calcium. It is possible that dif-ferences in intracellular amino acid concentrations may havecontributed to altered calcium signaling. Previously, we mea-sured amino acid amounts in cells with T1R3 knocked down

following amino acid withdrawal and subsequent amino acidrepletion (12). Intracellular concentrations dropped rapidlywhen extracellular amino acids were removed. In contrast toamino acid withdrawal, T1R3 receptor knockdown did notreduce intracellular amino acid content. Branched side chainamino acids in particular were similar. Thus, intracellularamino acids may have led to some of the calcium responses thatdiffered between receptor knockdown and amino acid-deprived cells. Finally, increased T1R3 expression in the aminoacid-deprived cells may also have contributed to the differencesnoted.

Our findings suggest that multiple types of nutrientresponses are linked with the M3R to support � cells during

FIGURE 6. Carbachol-stimulated mobilization of calcium from intracellular stores is unaltered in MIN6 cells deprived of amino acids. A, amino acid-deprived and control MIN6 cells were stimulated with 100 �M carbachol in calcium-free KRBH to assess release of calcium from intracellular stores. Data arenormalized mean peak 340/380 values � S.E. from three independent experiments, each in triplicate. B, mean peak 340/380 values � S.E. from data in A. C,amino acid-deprived and control MIN6 cells loaded with Fura-2/AM were subjected to the calcium depletion/repletion protocol in Fig. 2C. Data are normalizedmean 340/380 values � S.E. from three independent experiments, each in triplicate. D, mean peak 340/380 values � S.E. from data in C. *, p � 0.05; **, p � 0.046;***, p � 0.048; paired Student’s t test. E, expression of T1R3 and GAPDH mRNA. Results are expressed as mean � S.E. from three independent experiments. *,p � 0.001; paired Student’s t test. RFU, relative fluorescent units.




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nutrient stress. Beneficial effects of M3R activation in � cellshave been deduced from defects in M3R knockout mice andwere also revealed in studies of a designer Gq-coupled receptorengineered by incorporating mutations in M3R that renderedthe receptor unresponsive to ACh but selectively sensitive toactivation by the pharmacologically inert compound clozapine-N-oxide (13, 31–33). Chronic activation of the � cell Gq-cou-pled designer receptor in mice resulted in enhanced insulinrelease, decreased blood glucose concentrations, augmented �cell mass because of stimulation of � cell proliferation,increased insulin content, and amplified expression of severalgenes critical for � cell function (13, 33).

In addition to studies performed in mice, variations in thegene encoding M3R in humans are associated with a reducedacute insulin response and increased risk for early-onset type 2diabetes (34). Type 2 diabetes is characterized by hyperglyce-mia resulting from the inability of � cells to secrete sufficientinsulin to overcome peripheral insulin resistance (35). Theincreased demand on � cells to secrete insulin leads to � cellexhaustion, reduced � cell mass, and impaired insulin produc-tion (35, 36). The increase in M3R expression in amino acid-deprived cells provides independent support for the idea thatmodulating the expression of and/or signaling through � cellM3Rs enhances � cell function and protects against some typesof nutrient stress.

Acknowledgments—We thank Elliott Ross (Department of Pharma-cology) for suggestions, Michael Kalwat and other members of theCobb laboratory for comments on the manuscript, and Dionne Warefor administrative assistance.

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Marcy L. Guerra, Eric M. Wauson, Kathleen McGlynn and Melanie H. CobbAcid Signaling

Cells Is Enhanced by Impaired AminoβMuscarinic Control of MIN6 Pancreatic

doi: 10.1074/jbc.M114.565069 originally published online April 2, 20142014, 289:14370-14379.J. Biol. Chem.

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MuscarinicControlofMIN6Pancreatic …Signaling by the M3R also activates ERK1/2 in cells, most likely downstream of elevated intracellular calcium (12–14). ERK1/2 activation enhances