Glutamine-induced signaling pathways via amino acid receptors in enteroendocrine L cell lines

in Journal of Molecular Endocrinology

Correspondence should be addressed to T Tsuboi: takatsuboi@bio.c.u-tokyo.ac.jp

*(T Nakamura, K Harada and T Kamiya contributed equally to this work)

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Glucagon-like peptide-1 (GLP-1), secreted by gastrointestinal enteroendocrine L cells, induces insulin secretion and is important for glucose homeostasis. GLP-1 secretion is induced by various luminal nutrients, including amino acids. Intracellular Ca2+ and cAMP dynamics play an important role in GLP-1 secretion regulation; however, several aspects of the underlying mechanism of amino acid-induced GLP-1 secretion are not well characterized. We investigated the mechanisms underlying the L-glutamine-induced increase in Ca2+ and cAMP intracellular concentrations ([Ca2+]i and [cAMP]i, respectively) in murine enteroendocrine L cell line GLUTag cells. Application of L-glutamine to cells under low extracellular [Na+] conditions, which inhibited the function of the sodium-coupled L-glutamine transporter, did not induce an increase in [Ca2+]i. Application of G protein-coupled receptor family C group 6 member A and calcium-sensing receptor antagonist showed little effect on [Ca2+]i and [cAMP]i; however, taste receptor type 1 member 3 (TAS1R3) antagonist suppressed the increase in [cAMP]i. To elucidate the function of TAS1R3, which forms a heterodimeric umami receptor with taste receptor type 1 member 1 (TAS1R1), we generated TAS1R1 and TAS1R3 mutant GLUTag cells using the CRISPR/Cas9 system. TAS1R1 mutant GLUTag cells exhibited L-glutamine-induced increase in [cAMP]i, whereas some TAS1R3 mutant GLUTag cells did not exhibit L-glutamine-induced increase in [cAMP]i and GLP-1 secretion. These findings suggest that TAS1R3 is important for L-glutamine-induced increase in [cAMP]i and GLP-1 secretion. Thus, TAS1R3 may be coupled with Gs and related to cAMP regulation.

Supplementary Materials

    • Supplementary figure 1. Relationship between basal brightness of Fluo3 (A) or Flamindo2 (B) and the extent of response to 500 μM L-glutamine. Pearson’s correlation coefficient, R = 0.0655, P = 0.669, N = 45 (A) and R = −0.251, P = 0.198, N = 28 (B).
    • Supplementary figure 10. Sequences of off-target mutation candidate loci of TAS1R3-ΔC GLUTag cell lines. Off-target candidate sites are shown in gray. All sequences of candidate sites did not contain any insertions or deletions, and were consistent with mouse reference genome sequences GRCm38/mm10).
    • Supplementary figure 2. Response to higher concentration of L-glutamine. A, Typical time course of FI of Fluo3 after application of 500 μM and 10 mM L-glutamine. B, Peak amplitude calculated from the FI of Fluo3 by application of 500 μM and 10 mM L-glutamine; N ≥ 28 cells from three independent experiments; Welch’s t test. C, Typical time course of FI of Flamindo2 during application of 500 μM and 10 mM L-glutamine. D, Area under curve (AUC) calculated from the FI of Flamindo2 by application of 500 μM and 10 mM L-glutamine; N ≥ 10 cells from three independent experiments; Welch’s t test. Data presented as mean ± standard error of the mean (SEM). **** p < 0.0001.
    • Supplementary figure 3. Response to positive control stimulation, and examination of the specificity of antagonists used with L-glutamine. A, Typical time course of FI of Fluo3 during application of 500 μM glutamine, high [K+] solution, high [K+] solution with 3 μM NPS-2143, 3 μM NPS-2143 alone, and vehicle (DMSO). B, Peak amplitude calculated from the FI of Fluo3 by application of 500 μM L-glutamine, high [K+] solution, high [K+] solution with 3 μM NPS-2143, 3 μM NPS-2143 alone, and vehicle; N ≥ 25 cells from three independent experiments; One-way ANOVA with Tukey’s multiple comparison test. C, Typical time course of FI of Flamindo2 during application of 500 μM Lglutamine, 10 μM forskolin (fsk), 10 μM fsk with 3 mM lactisole, 3 mM lactisole alone, and vehicle (DMSO). D, Area under the curve (AUC) calculated from the FI of Flamindo2 by application of 500 μM L-glutamine, 10 μM fsk, 10 μM fsk with 3 mM lactisole, 3 mM lactisole alone, and vehicle; N ≥ 12 cells from three independent experiments; One-way ANOVA with Tukey’s multiple comparison test. Data pertaining to 500 μM glutamine are from Supplementary figure 2. Data presented as mean ± standard error of the mean (SEM). N.S., not significant. ****p < 0.0001.
    • Supplementary figure 4. Expression of amino acid-sensitive GPCRs and the role of G proteins in L-glutamine-induced increase in [Ca2+]i and [cAMP]i levels. A, RT-PCR analysis for Casr, Gprc6a, Tas1r1, and Tas1r3 in GLUTag cells. Gapdh was utilized as a positive control of RT-PCR. N = 3 experiments. B, Typical time course of fluorescence intensity (FI) of Fluo3 during application of 500 μM L-glutamine after treatment with 25 μM BIM-46187. C, Peak amplitude calculated from the FI of Fluo3 by application of 500 μM L-glutamine after treatment with 25 μM BIM-46187; N ≥ 26 cells; Welch’s t test. D, Typical time course of FI of Flamindo2 during application of 500 μM L-glutamine after treatment with 25 μM BIM-46187. E, Area under the curve (AUC) calculated from the FI of Flamindo2 by application of 500 μM L-glutamine after treatment with 25 μM BIM-46187; N = 29 cells; Welch’s t test. Data presented as mean ± standard error of the mean (SEM). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
    • Supplementary figure 5. Examination of the expression of EGFP in CRISPR/Cas9-based cells. A, PCR to amplify EGFP fragments using mutant GLUTag cell lines. Almost 2000 copies were added into PCR mixture. pSpCas9(BB)-2A-GFP plasmid was utilized as a positive control. EGFP bands were detected in TAS1R1-ΔC #1, #2, #3 and TAS1R3-ΔC #1. gDNA; genome DNA. B, Microscopic images of the cells showing EGFP integrated in the genomes. Parental cells (without transfection), pEGFP-C1-expressing cells, and pSpCas9-GFP-expressing cells were observed as negative and positive controls. Scale bar, 100 μm.
    • Supplementary figure 6. Genome sequence of additional TAS1R1-ΔC GLUTag cell lines. PAM, protospacer adjacent motif sequence; sgRNA, single guide RNA; bp, base pairs; del, deletion; ins, insertion.
    • Supplementary figure 7. Genome sequence of additional TAS1R3-ΔC GLUTag cell lines. PAM, protospacer adjacent motif sequence; sgRNA, single guide RNA; bp, base pairs; del, deletion; ins, insertion.
    • Supplementary figure 8. Raw traces of Fluo3 and Flamindo2 in CRISPR/Cas9-based cells. A-I, Typical time course of FI of Fluo3 in Ctrl #1–3 (A–C), TAS1R1-ΔC #1–3 (D–F), TAS1R3-ΔC #1–3 (G–I) cells during application of 500 μM L-glutamine. J–R, Typical time course of FI of Flamindo2 in Ctrl #1–3 (J–L), TAS1R1-ΔC #1–3 (M–O), TAS1R3-ΔC #1–3 (P–R) cells during application of 500 μM L-glutamine.
    • Supplementary figure 9. Sequences of off-target mutation candidate loci of TAS1R1-ΔC GLUTag cell lines. Off-target candidate sites are shown in gray. All sequences of candidate sites did not contain any insertions or deletions, and were consistent with mouse reference genome sequences (GRCm38/mm10).
    • Information of PCR primers

 

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