Development of a red fluorescent protein-based cGMP indicator applicable for live-cell imaging

Cyclic guanosine 3′, 5′-monophosphate (cGMP) is a second messenger that regulates a variety of physiological processes. Here, we develop a red fluorescent protein-based cGMP indicator, “Red cGull”. The fluorescence intensity of Red cGull increase more than sixfold in response to cGMP. The features of this indicator include an EC50 of 0.33 μM for cGMP, an excitation and emission peak at 567 nm and 591 nm, respectively. Live-cell imaging analysis reveal the utility of Red cGull for dual-colour imaging and its ability to be used in conjunction with optogenetics tools. Using enteroendocrine cell lines, Red cGull detects an increase in cGMP following the application of l-arginine. An increase in intracellular cGMP is found to be inhibited by Ca2+, and l-arginine-mediated hormone secretion is not potentiated. We propose that Red cGull will facilitate future research in cell signalling in relation to cGMP and its interplay with other signalling molecules.

C yclic guanosine 3′, 5′-monophosphate (cGMP) is an important second messenger involved in a variety of physiological functions such as smooth muscle relaxation and phototransduction 1 . Intracellular cGMP concentration is strictly regulated via production by cytosolic/soluble guanylyl cyclase (sGC) or membrane-bound/particulate guanylyl cyclase and through degradation by phosphodiesterase 1 . The complexity of intracellular cGMP dynamics depends on the balance of the activation and expression level of these proteins. Thus, revealing the precise spatiotemporal regulation mechanisms of intracellular cGMP dynamics, particularly its interactions with other signalling molecules such as Ca 2+ , is an important research goal.
sGC is the only conclusively proven receptor for nitric oxide (NO), a signalling molecule produced by the enzyme nitric oxide synthase (NOS) from the amino acid L-arginine 2 . The NO/cGMP signalling pathway is associated with several cell functions, including relaxation of vascular muscles and neurotransmission 3 . Furthermore, evidence suggests that this pathway is involved in exocytotic functions through the activation of some targeted proteins, such as phosphodiesterase, protein kinases, or ionic channels; NO donors, nitrites, and cGMP analogues potentiate glucose-induced insulin secretion from pancreatic β cells [4][5][6] . Glucagon-like peptide-1 (GLP-1), a gut hormone secreted from enteroendocrine L-cells, plays a role in potentiating glucosedependent insulin secretion and reducing appetite 7,8 . Glucose, several types of amino acids, and fatty acids have all been identified as secretagogues of GLP-1 [9][10][11] . Recently, it was reported that administration of L-arginine enhances the secretion of GLP-1 and peptide YY (PYY), another gut hormone secreted from L-cells, in both rodents and humans 12,13 . However, the involvement of the NO/cGMP signalling pathway in L-arginine-induced GLP-1 secretion remains to be verified.
Genetically encoded fluorescent protein (FP) indicators based on variants of the green fluorescent protein (GFP) are powerful tools for imaging intracellular cGMP dynamics. Most currently available cGMP indicators are Förster resonance energy transfer (FRET)-based indicators 14,15 . Since FRET imaging requires the acquisition of emitted light from the donor and acceptor proteins, this process has limited flexibility in multicolour imaging. Single FP-based indicators can be used to overcome this problem; thus, many single FP-based indicators have been developed to detect signalling molecules (Ca 2+ and cAMP) and metabolites (glucose, pyruvate or lactate), including the GFP-based cGMP indicator that we and another group previously developed [16][17][18][19][20] .
Optogenetic methods are also useful for modulating intracellular signalling molecules by light. Previously, blue light-activated guanylyl cyclase (bacterial light-activated guanylyl cyclase: BlgC) was purified and engineered onto the base of bacterial light-activated adenylyl cyclase (BlaC) from the Beggiatoa sp. PS genome; it has been proposed for use in in vitro experiments involving mammalian cells after human-codon optimisation 21,22 . To extend the colour palette of single FP-based cGMP indicators and enable integration into optogenetic experiments, a nongreen sensor is required.
In the present study, we demonstrate the development of a red single FP (mApple)-based cGMP indicator, namely Red cGull (Red cGMP visualising fluorescent protein). We show that live-cell imaging can be conducted using Red cGull under single and dualcolour acquisition and that this indicator can be implemented in optogenetics. We also reveal the Ca 2+ and cGMP dynamics in L-arginine-induced GLP-1 secretion from enteroendocrine cells.

Results
Red cGull is a single FP-based cGMP indicator. The green FPbased cGMP indicator, Green cGull, was originally reported by our group. It was designed by inserting the cGMP-binding domain of cGMP-specific mouse phosphodiesterase 5α (mPDE5α) near the chromophore of a GFP variant, Citrine, with linker sequences derived from the leucine zipper sequences [22][23][24] . Here, we inserted the same cGMP-binding domain into a red FP, mApple, to develop a prototype of a red cGMP indicator ( Fig. 1a and Supplementary Fig. 1a). Based on our previous work, we learned that mutations in linker peptide sequences largely affect the dynamic range (F/F 0 ) of FI of indicators in response to target molecules. Therefore, we constructed numerous variants of the indicator with different linker lengths and amino acid sequences ( Supplementary Fig. 1a, b). Following the screening, we identified the mutant with the largest FI in the presence of 10 μM cGMP, which we named Red cGull ( Fig. 1b and Supplementary Fig. 2).
Red cGull shows a sixfold FI increase in response to cGMP in vitro. We first analysed the in vitro properties of Red cGull using purified recombinant proteins from E. coli. According to fluorescence spectra, Red cGull had excitation and emission peaks at 567 and 591 nm, respectively (Fig. 2a). The FI of Red cGull increased 6.7-fold in the presence of 10 μM cGMP, which is a saturating dose. The absorption spectra of Red cGull showed a higher peak near 450 nm in absence of cGMP, and a higher peak near 570 nm in the presence of 100 µM cGMP (Fig. 2b). Extinction coefficients were calculated from the peak at 562 nm ( Table 1). The quantum yield of Red cGull increased 1.5 times in the cGMP saturated state (Table 1). For negative control, Red cGull nega ( Supplementary Fig. 1), which showed little change in fluorescence intensity in the presence of cGMP ( Supplementary  Fig. 3), was selected, and extinction coefficient and quantum yield were displayed in Table 1.
We also examined the dose-response relationship of Red cGull for cGMP and calculated the half-maximal effective concentration (EC 50 ) value by fitting a four-parameter logistic curve. The EC 50 value of Red cGull for cGMP was 0.33 μM; in contrast, Red cGull exhibited a negligible response to a structurally similar substance, cAMP (Fig. 2c). In addition, the response of Red cGull was reversible, and the FI of Red cGull reached 80% of its maximum within 5 s ( Supplementary Fig. 4). Taken together, these results indicate that Red cGull detects cGMP and shows a consequent increase in FI in vitro. a Diagrams of mApple, mPDE5α and Red cGull. mPDE5α contains GAF domains for cGMP binding and HD domains for phosphodiesterase activity. See also Fig. S1. b Three-dimensional schematic images of Red cGull unbound (left) and bound (right) to cGMP. Images were created using structural data for mCherry (PDB_4ZIO) and PDE5α (cGMP-unbound: PDB_3MF0; cGMP-bound: PDB_2K31).
Red cGull monitors intracellular cGMP dynamics. We next validated the utility of Red cGull in live cells. We expressed Red cGull in the mouse enteroendocrine L cell line GLUTag and human cervical epithelial carcinoma cell line HeLa, and then applied cGMP-inducing stimuli, SNAP (NO donor) or 8-Br-cGMP (a membrane-permeable cGMP analogue), to monitor the intracellular cGMP dynamics in modified Ringer's buffer (RB). Application of various concentrations of SNAP or 8-Br-cGMP elicited increases in the FI of Red cGull in each cell line (Fig. 3a, b and Supplementary Fig. 5a, b). For SNAP application, we applied 6 to 600 µM SNAP to GLUTag cells. About 6 µM SNAP induced a small increase in FI, and 60 µM SNAP induced a large increase of FI as 600 µM SNAP but decreased over time. For 8-Br cGMP application, we applied 0.5 to 2 mM 8-Br-cGMP to HeLa cells. About 0.5 mM 8-Br-cGMP did not induce changes in FI, while 1 mM induced a comparable increase as 2 mM 8-Br-cGMP. For validation of those response, we used Red cGull nega ( Supplementary Fig. 5c, d) and δ-FlincG, a previously developed fluorescent protein-based cGMP indicator (means ± standard deviation (s.d.), 113.7 ± 8.3, Supplementary Fig. 6a, c) 20 . Ca 2+ is an important second messenger and the interplay between Ca 2+ and cGMP is crucial for many physiological events such as smooth muscle relaxation and intestinal cell proliferation 25 . Given the advantage of single FP-based indicators, which are applicable to dual-colour imaging, we attempted to visualise the changes in both intracellular cGMP and Ca 2+ concentrations ([cGMP] i and [Ca 2+ ] i , respectively) by using Red cGull and the green Ca 2+ -sensitive dye Fluo4 in single cells. Application of  The quantum yields were measured by the absolute photoluminescence quantum yield measurement system (Hamamatsu Photonics, C9920-02). Data were shown as means ± standard deviation (n = 3). The extinction coefficients were calculated using absorbance at 562 nm (Fig. 2b). Data were shown as means ± standard deviation (n = 4). SNAP triggered a transient increase in the FI of both Red cGull and Fluo4 in GLUTag cells (Fig. 3c). These results suggest that Red cGull can be used to monitor intracellular cGMP dynamics in different types of cells and that it is applicable to dual-colour imaging for investigation of the interplay among several signalling molecules.
Red cGull detects cGMP produced by photoactivated guanylyl cyclase. To examine whether Red cGull was able to employ in conjunction with optogenetic tools, we coexpressed BlgC and Red cGull in GLUTag cells 21 . By intermittent stimulation with a 1.2 mW/cm 2 blue light laser (488 nm), the FI of Red cGull was shown to rapidly increase after each stimulus in coexpressing cells (Fig. 4a). In contrast, in a control experiment, blue light laser stimulation itself induced only minor increases in the FI of cells expressing Red cGull and the vector only (Fig. 4b). These results suggest that Red cGull detects cGMP levels produced by photoactivated guanylyl cyclase.
L-arginine induces cGMP production in the enteroendocrine cell line STC-1. As previous studies have shown that L-arginine potentiates the secretion of GLP-1 in rodents and humans 12,13 , we investigated whether cGMP plays a role in the secretion of gut hormones. First, we used real-time PCR to analyse the mRNA expression of sGC subunits in STC-1 cells, which secrete a variety of gut hormones, including GLP-1, similar to native enteroendocrine cells and are routinely used in in vitro experiments ( Fig. 5a) 26 . Real-time PCR analysis revealed mRNA expression of sGC α1 and β1 subunits, which are physiologically functional subunits ( Fig. 5a) 27 . Next, we examined the signalling cascades induced by L-arginine in STC-1 cells. Application of L-arginine to Red cGull-expressing STC-1 cells induced an increase in the FI of Red cGull (means ± s.d., 118.4 ± 10.3, p < 0.0001, Fig. 5b, d, means ± s.d., 112.7 ± 5.9, p < 0.0001, Fig. 5c,). We also applied Larginine to δ-FlincG-expressing STC-1 cells, and observed the increase of FI as well (means ± s.d., 118.5 ± 12.8, p < 0.0001, Supplementary Fig. 6b, c) 20 . L-NAME (a NOS inhibitor) and LY-83583 (a sGC inhibitor) inhibited the L-arginine-induced FI increase of Red cGull, suggesting that cGMP was produced via NOS and sGC activation (L-NAME: means ± s.d., 110.4 ± 5.3, p = 0.0419, Fig. 5c, LY-83583: means ± s.d., 112.7 ± 4.2, p = 0.0066, Fig. 5d). In a previous study, L-arginine was also sensed by nutrient-sensing receptors including the calciumsensing receptor (CaSR), G protein-coupled receptor family C subtype A (GPRC6A), and taste  Supplementary Fig. 7c). These results suggest that CaSR and Gq protein play primary roles in L-arginine-induced Ca 2+ signalling. We also explored the interplay between cGMP and Ca 2+ in STC-1 cells. Inhibition of G q protein by YM-254890 increased the FI of Red cGull (means ± s.d., 112.6 ± 5.7, p = 0.0005, Fig. 6a Fig. 8).

Discussion
Based on our previous method for establishing single FP-based indicators, we developed a single red FP-based cGMP indicator, Red cGull, for use in live-cell imaging. Expanding the colour palette of single FP indicators is important to the improvement of intracellular imaging, taking such imaging from a monochromatic to a multichromatic endeavour. Therefore, red single FP indicators have previously been developed, as exemplified by Ca 2+ , ATP, cAMP and glucose 17,[29][30][31] . However, until now, a red single FP-based indicator for cGMP had not been developed. Red cGull overcomes the limitations of cGMP imaging and is usable in conjunction with optogenetic tools and enables dual-colour imaging with different molecules in a single cell.   The response of Red cGull was 7.7 (F/F 0 ) at the end of the sitedirected random mutation screening process ( Supplementary  Fig. 1), which was higher than the response of Red cGull after purification (Fig. 2a). We think that the response of the purified protein was more reliable because they are free of potential contaminants. Thus, we finally defined the dynamic range of Red cGull as 6.7 (F/F 0 ) according to the emission spectra of purified recombinant protein data (Fig. 2a). It has been reported that, in single red FP-based indicators based on mApple, such as R-GECO, the protonated chromophore without fluorescence absorbs near 450 nm and the deprotonated chromophore with fluorescence absorbs near 570 nm 32 . Moreover, we found that change in quantum yield after the application of cGMP. Therefore, both changes respond to cGMP in Red cGull induces its FI increase. We found that the EC 50 of Red cGull was 0.33 μM, which was close to the widely used FRET-based cGMP indicator 15 . The physiological level of cGMP is believed to be <5 μM; thus, Red cGull should be applicable at the physiological cGMP level in many cells 15 . However, because certain cells, such as cardiomyocytes or stellate ganglion neurons, contain low cGMP concentrations (nM range), more sensitive indicators with higher affinities to cGMP might be required to expand the usability of the indicator 33 .
To examine the utility of Red cGull in live-cell imaging, we expressed Red cGull in various cell lines. Both the NO donor SNAP and the cGMP analogue 8-Br-cGMP induced an increase in FI; however, their kinetics seemed to be different. This difference would be due to the difference in the properties of those compounds. 8-Br-cGMP is a phosphodiesterase-resistant cGMP analogue and showed monotonically increasing cGMP changes over time, whereas SNAP produced cGMP via the activation of sGC; the produced cGMP was degraded by phosphodiesterase.

During dual-colour imaging in GLUTag cells, SNAP triggered increases in both [Ca 2+ ] i and [cGMP] i . Such SNAP-dependent [cGMP] i and [Ca 2+
] i increase may reflect the cGMP-dependent activation of cyclic nucleotide-gated channels, which is one signalling pathway previously reported in relation to a cGMPdependent [Ca 2+ ] i increase 34 . The FI of Red cGull was saturated at~2.5-fold in live-cell imaging, which is lower than its dynamic range (6.7 (F/F 0 )) obtained via in vitro spectrometry with purified Red cGull proteins. This difference may be explained by a certain basal level of cGMP in cells 15 .
The ability to monitor and manipulate intended signal transduction is becoming essential to the study of cell biology and especially neuroscience. By using Red cGull and photoactivated guanylyl cyclase, i.e. BlgC, we were able to manipulate cGMP production and then monitor cGMP in real-time. With repeated blue light stimuli, we showed that only a short duration of exposure (0.2 s) was sufficient to produce cGMP. We believe that longer stimulation would produce more cGMP, which may degrade quickly after they are produced; hence, the responses appear to be similar to that of short exposures. We think that the combination of cGMP imaging and optogenetic techniques has the potential to expand the field and help provide answers to important questions.
L-arginine, a conditionally essential amino acid, is contained in various foods and produced in cellular metabolism. L-arginine is transported via cationic amino acid transporters and used to produce NO, which in turn promotes the generation of cGMP by sGC 35 . sGC is a heterodimer with α and β subunits. Within two isoforms, α1 and β1 are physiologically functional in most tissues, including the small intestine 27 . Although our data show that Larginine potentiates GLP-1 secretion in STC-1 cells, which is consistent with previous in vivo experiments, the cGMP produced by L-arginine application itself did not stimulate GLP-1 secretion 12,13 . In pancreatic β cells, cGMP derived from NO stimulates insulin secretion; however, this effect reportedly occurs only when NO exists in low concentrations (<50 nM); when NO concentrations are high (>80 nM), insulin secretion is instead somewhat inhibited 6 . Because a bidirectional effect of NO exists, 10 mM of L-arginine might not be a suitable concentration to activate the NO/cGMP signalling pathway and induce hormone secretion in STC-1 cells. L-arginine is also sensed by all three promiscuous amino acid sensing receptors. In our work, only a CaSR antagonist, Calhex231, suppressed the L-arginine-induced Ca 2+ increase; however, it was reported that GPRC6A antagonist, Calindol, also functions as an allosteric modulator of CaSR 36 . Therefore, we consider whether the inhibitory effect on GPRC6A or activation effect on CaSR may have been counterbalanced and whether Calindol had no effect on Ca 2+ signalling. Thus, it is difficult to judge whether GPRC6A is relevant in L-arginine signalling. Clemmesen et al. used GPRC6A-knockout mice and determined that GPRC6A is not required in L-arginine-mediated GLP-1 secretion 37 . Further experiments, such as those including specifically targeted antagonists or knockout mice, will be required to reveal the mechanism of L-arginine-mediated GLP-1 secretion in the future.

Conclusion
In conclusion, we successfully developed the red single FP-based cGMP indicator, which we have named Red cGull. In vitro analysis showed that the FI of Red cGull increased more than sixfold in the presence of cGMP. Red cGull is applicable for livecell imaging of cGMP, including dual cGMP/Ca 2+ imaging and for cGMP imaging in conjunction with optogenetic stimulation. Overall, our results demonstrate that Red cGull overcomes the limited utility of FRET-based or green FP-based indicators to accommodate existing excitation and emission wavelength windows. The accessibility of our technology will enable researchers in a range of disciplines to investigate the NO/cGMP signalling pathway, e.g., in health and disease, in not only enteroendocrine cells but also other cells, tissues and animal models.
Plasmid construction. The DNA fragment of a red FP variant, mApple, was created by DNA synthesis from Integrated DNA Technologies (Coralville, IA, USA). 29 Subsequently, mApple was modified by PCR to insert SacII and EcoRI restriction enzyme sites at a position between A150 and V151 before being cloned into the pRSET-A vector at the BamHI/HindIII site, as previously described 17 . During this step, the hyper acidic region of a fragment of mouse amyloid precursor protein (APP; NM_001198823.1, amino acids 190-286) was amplified from adult mouse whole brain mRNA via RT-PCR and then fused to the N-terminus of mApple to improve the solubility of the protein under bacterial expression 38 . The cDNA for the cGMP-binding GAFa domain of mouse phosphodiesterase 5α (mPDE5α, NM_153422.2, amino acids 164-298) was amplified from adult mouse whole brain mRNA using RT-PCR and then inserted into the SacII/EcoRI sites of the modified mApple in pRSET-A (i.e. the Red cGull prototype) 15,24 . To expand the dynamic range of the indicator by linker length optimisation, leucine zipper sequences of various lengths were inserted between mApple and the cGMPbinding domain 39 . The mutant with the greatest fluorescence intensity was selected for further optimisation. PCR was performed using two sets of primers, including NNK and MNN to introduce random mutations at one position in the linker amino acid sequences. In one screening,~50 random mutations were produced; the mutation with the greatest dynamic range was selected as the next template for sitedirected random PCR. After the repetitive screening, a mutant that resulted in the maximum dynamic range by cGMP was produced; this mutant was named Red cGull. For expression in mammalian cells, Red cGull was subcloned into the pcDNA3.1(−) vector (Thermo Fisher Scientific, Waltham, MA, USA) at the BamHI/HindIII site. To generate photoactivated guanylyl cyclase (i.e. BlgC), the DNA sequence of bPAC was amplified by PCR from pGEM-HE-h_bPAC_cmyc (Addgene, Watertown, MA, USA; #28134) and three point mutations (i.e. K197E, D265K and T267G) were introduced to facilitate guanylyl cyclase activity based on a previous study 21 . The resultant sequence was inserted into the pEGFP-C1 vector for live-cell imaging. To generate lentivirus, Red cGull was subcloned into the backbone vector plasmid, CSII-EF-MCS (RIKEN RBC DNA BANK, #RDB04378), at the NotI/BamHI site.
Protein expression and purification. For protein expression, pRSET-A with APPfused Red cGull was transformed into Escherichia coli JM109 (DE3) (Promega, Madison, WI, USA) and cultured in LB medium with 50 mg/L ampicillin (FUJI-FILM Wako Pure Chemical Corporation) at 20°C for 4 days. The cells were then centrifuged at 7000 rpm and 4°C for 10 min. The resultant pellets were suspended in phosphate-buffered saline (PBS) with 40 µg/mL lysozyme (FUJIFILM Wako Pure Chemical Corporation) and lysed by freeze-thawing and ultrasonic homogenisation. Subsequently, nickel-nitrilotriacetic acid agarose beads (QIAGEN, Venlo, Netherlands) were added to the recovered supernatant and absorbed via rotation at 4°C for 3-6 h. The beads were then recovered by centrifugation and resuspended in PBS. The supernatant was added to a filtered column, washed three times with PBS, washed three times with 3 mL of 10 mM imidazole/PBS, and then eluted using 5 mL of 300 mM imidazole/PBS. To remove imidazole, 1 mL of elution was added to a PD-10 filtration column (GE Healthcare, Buckinghamshire, UK) in HEPES buffer (150 mM KCl and 50 mM HEPES). This purified protein was analysed to measure excitation and emission spectra, a dose-response curve, and absorption spectra.
In vitro spectrometry. Excitation (for 595 nm) and emission (at 550 nm) spectra were measured in the absence or presence of 10 µM cGMP in 5 µM of purified protein/HEPES buffer using a fluorescence spectrophotometer (F-2500; Hitachi, Tokyo, Japan). The absorption spectra of Red cGull were measured using a UV spectrometer (UV-1800; Shimadzu, Kyoto, Japan). Absorption spectra was corrected using the absorbance at 650 nm as the background. The quantum yields were measured by the absolute photoluminescence quantum yield measurement system (Hamamatsu Photonics K.K., Shizuoka, Japan, C9920-02: excitation wavelength: 550 nm). To generate a dose-response curve, the fluorescence intensity (FI) of the purified proteins, diluted to 1 µM with HEPES, was measured in the presence or absence of cGMP or cAMP. The EC 50 was calculated using the equation for the four-parameter logistics curve in the Rodbard mode of ImageJ's curve fitter function (National Institutes of Health, Bethesda, MD, USA). The normalised dynamic range for cAMP was converted based on the maximum and minimum parameters of cGMP measurements; a logarithmic approximation line was then drawn.
Lentivirus production. For lentivirus production, the following three transfection plasmids were mixed in 15 mL tubes: 10 μg of pCAG-HIVgp (RIKEN BRC DNA BANK, #RDB04394), 10 μg of pCMV-VSV-G-RSV-Rev (RIKEN RBC DNA BANK, #RDB04393), and 17 μg of CSII-EF-Red cGull. DNA mixtures were then diluted with 36 μL of Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) and incubated for 20 min at room temperature. HEK293T cells were seeded onto 10 cm culture dishes at a density of 5 × 10 5 cells and then the transfection mixture was transferred to the cells before they were incubated at 37°C under a 5% CO 2 atmosphere. Viruses were harvested at 48 and 96 h posttransfection. Viral supernatants were centrifuged at 780×g and 4°C for 10 min and then the supernatant was filtered through a 0.45 μm filter (Merck Millipore, Burlington, MA, USA) to recover the virus.
Plasmid transfection and lentivirus infection. For live-cell imaging, cells were trypsinised and plated onto poly-L-lysine (PLL; Sigma-Aldrich)-coated glass coverslips in 35-mm dishes. Two days after plating, the cells were transfected with plasmids (1.5 μg for single-wavelength imaging and 0.5 μg each for two-wavelength imaging with photoactivated protein) using 3 μL of Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. Four hours after transfection, the medium was changed, and the cells were cultured at 30-32°C for 2 days until imaging was conducted. For lentivirus infection, STC-1 cells were plated in PLL-coated dishes (35 mm). Two days after plating, 1 mL of lentivirus and 25 μg/mg of polybrene (Nacalai Tesque) were mixed and added to the cell culture with 1 mL of DMEM (high glucose). The cells were then incubated at 37°C under a 5% CO 2 atmosphere for 2 days. On the day before the imaging experiment, the high-glucose DMEM culture medium was changed to a low-glucose DMEM medium, and cells were cultured at 30-32°C.
RNA isolation and real-time PCR. Total RNA from STC-1 cells and mouse tissues (heart, brain, and testis) was isolated using RNeasy Mini Kit (QIAGEN). After DNase treatment using RNase-Free DNase Set (QIAGEN), cDNA was synthesised using High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific). Synthesised cDNA was amplified using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan). The reaction was performed using Thermal Cycler Dice (TAKARA BIO Inc.) in a two-step reaction (95°C for 1 min for initial denaturation, 95°C 15 s, 60°C 30 s; 40 cycles). Primer sequences used in real-time PCR analysis are shown in Supplementary Table 1. The experiments were repeated three times. GAPDH was used as the reference gene in each template, and the relative expression level of the target gene was calculated using the ΔΔCt method. The expression level of the tissue was then used as a positive control, normalised to 1 for each gene, and the expression levels of the STC-1 cells were calculated.
Enzyme-linked immunosorbent assay. STC-1 cells were plated in 24 well plates at 1.0 × 10 5 cells per well. After 24 h, the cultured medium was changed from high-to low-glucose DMEM. Two days after plating, cells were washed twice with RB containing 2.2 mM glucose and 0.5% (w/v) bovine serum albumin (BSA; FUJIFILM Wako Pure Chemical Institute). Subsequently, vehicle solution (dimethyl sulfoxide), 10 mM L-arginine, or 10 mM L-arginine with either 1 μM LY-83583 or 250 nM YM-254890 in RB containing 2.2 mM glucose and 0.5% BSA were applied to the cells and incubated for 2 h at 37°C under a 5% CO 2 atmosphere. After incubation, cell supernatant was collected into collection tubes supplemented with 60-KIU aprotinin (FUJIFILM Wako Pure Chemical Institute) and 34 μg/mL diprotin A (Peptide Institute, Inc., Osaka, Japan). After centrifugation at 1000×g and 4°C for 10 min, the supernatant was diluted in RB containing 2.2 mM glucose and 0.5% (w/v) BSA and then used for analysis with a GLP-1 Active Form Assay Kit (#27784; IBL, Gunma, Japan) and microplate reader (Varioskan LUX; Thermo Fisher Scientific) according to the manufacturer's protocols. Changes were calculated relative to vehicle-treated GLP-1 secretion.
Statistics and reproducibility. Final data are shown as means ± standard deviations from n cells of more than three independent experiments, as indicated in the figure legends. When using bar charts, individual data points were overlaid. p value of 0.05 was considered significant. p values were stated in the graph legend. Statistical analysis in real-time PCR was performed using a two-tailed Welch's t-test. Normality was evaluated by Shapiro-Wilk test, and a multiple comparison test, whether parametric or nonparametric, was selected based on the normality test. Statistical analysis in imaging acquisition were performed using Kruskal-Wallis test with Dunn's multiple comparison. Statistical analysis in ELISA was performed using a One-way ANOVA with Tukey's multiple comparison test. These analyses were conducted in GraphPad Prism 6 software (GraphPad Software, La Jolla, CA, USA).
Reporting summary. Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper.