The camp Sensor Epac2 Is a Direct Target of Antidiabetic Sulfonylurea Drugs

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1 Supporting Online Material for The camp Sensor Epac2 Is a Direct Target of Antidiabetic Sulfonylurea Drugs Chang-Liang Zhang, Megumi Katoh, Tadao Shibasaki, Kohtaro Minami, Yasuhiro Sunaga, Harumi Takahashi, Norihide Yokoi, Masahiro Iwasaki, Takashi Miki, Susumu Seino* *To whom correspondence should be addressed. seino@med.kobe-u.ac.jp This PDF file includes: Published 31 July 29, Science 325, 67 (29) DOI: /science Materials and Methods Figs. S1 to S7 References

2 Supporting Online Material (Zhang et al.) Contents 1. Materials and Methods 2. References (S1-S7) 3. Supplementary Figure Legends 4. Supplementary Figures (S1-S7) 1. Materials and Methods Reagents Glibenclamide was purchased from ALEXIS (San Diego, CA). Tolbutamide, chlorpropamide, acetohexamide, glipizide, nateglinide, repaglinide, and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from SIGMA (St. Louis, MO). Gliclazide was purchased from LKT Laboratories Inc. (St. Paul, MN). [ 3 H]glibenclamide was purchased from Perkin Elmer (Waltham, MA). Mice Epac2 -/- mice were generated as described previously (S1). Wild-type C57BL/6 mice were used as control. All animal experiments were performed in accordance with the guidelines of the Animal Care Committee of Kobe University. Epac2-deficient clonal pancreatic β-cells Epac2-deficient, mouse clonal pancreatic β-cells were established as described previously (S1). Construction of FRET sensors 1

3 Wild-type mouse Epac2 (Epac2 wt) and Epac2 mutant [Epac2 (G114E, G422D)] (S2) were fused amino-terminally to enhanced cyan fluorescent protein (ECFP) and carboxyl-terminally to enhanced yellow fluorescent protein (EYFP). Cell culture and transfection MIN6 and COS-1 cells were grown in Dulbecco s modified Eagle s medium (DMEM) containing 1% heat-inactivated fetal bovine serum and maintained in a humidified incubator with 95% air and 5% CO 2 at 37 C. Two days before FRET measurements, cells were transiently transfected with plasmids encoding FRET sensors using FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals, Basel, Switzerland) according to the manufacturer s instructions. One day before FRET measurements, cells were reseeded in uncoated 25-mm glass-bottom dishes. Imaging Forty-eight hours after transfection with FRET sensors, the cells were subjected to imaging experiments. The growth medium was replaced with HEPES-KRB buffer containing mm NaCl, 4.7 mm KCl, 1.2 mm KH 2 PO 4, 1.2 mm MgSO 4, 2.5 mm CaCl 2, 5. mm NaHCO 3, 2.8 mm glucose, 2 mm HEPES (ph 7.4), and.2% BSA. The cells were observed at room temperature using confocal laser scanning microscopy (FV1, Olympus, Tokyo, Japan) equipped with an UPlanSApo objective lens (1 x oil/1.4 NA). The cells were excited by 44 nm LD laser (FV5-LDPSU, Olympus) with.5% output power. For kinetic FRET analysis, dual-emission imaging was performed using two emission filters (48DF3 for ECFP and 535DF25 for EYFP) in intervals of 5 sec. FRET was monitored as the emission ratio of EYFP to ECFP (YFP/CFP ratio). All image and FRET monitoring was controlled by FLUOVIEW software (Olympus). The YFP/CFP ratio was normalized to the R to describe FRET efficiency changes (FRET change = R/R ) where R is the YFP/CFP ratio at the stimulated time point. 2

4 Sulfonylurea binding experiments COS-1 cells were transfected with mouse Epac2 or human SUR1 cdna. Two days after transfection, the cells were collected, washed twice, and resuspended with assay buffer containing 119 mm NaCl, 4.7 mm KCl, 2.5 mm CaCl 2, 1.2 mm KH 2 PO 4, 1.2 mm MgSO 4, 5. mm NaHCO 3, and 2. mm HEPES (ph 7.4). Each aliquot (4 μl) containing x 1 5 cells was incubated for 1 h at room temperature with [ 3 H]glibenclamide in the absence or presence of unlabeled glibenclamide or tolbutamide at various concentrations. Bound [ 3 H]glibenclamide was separated from free [ 3 H]glibenclamide by rapid vacuum filtration through Whatmann GF/C filters (Whatmann International, Maidstone, U.K.). The filters were washed three times with 4 ml of ice-cold buffer (shown above) and the radioactivity was determined by liquid scintillation counter. Pull-down assay for GTP-Rap1 Pull-down assay for GTP-Rap1 was performed as described previously (S1). Thirty minutes after preincubation, the cells were treated with HEPES-KRB buffer containing 2.8 mm glucose for 15 min in the absence or presence of various stimuli as described in Fig. 3 and Fig. S5, S6. The cellular lysates were incubated with GST-RalGDS-RID (S3) immobilized on glutathione-resin (SIGMA). After incubation at 4 C for 9 min, the affinity-purified protein complexes were subjected to SDS-PAGE, followed by immunoblot detection with anti-rap1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Signals were quantified by Image Scanner (Amersham Pharmacia Biotech). Adenovirus-mediated gene transfer Adenovirus-mediated gene transfer was performed as described previously (S1). Briefly, recombinant adenovirus carrying β-galactosidase (Ad-β Gal) or Epac2 3

5 (Ad-Epac2) was generated according to the manufacturer's instructions (Stratagene, CA). Epac2-deficient, mouse clonal β-cells were infected with Ad-β Gal or Ad-Epac2. After 3-day culture, the infected cells were preincubated with 2.8 mm glucose and then incubated with 2.8 mm glucose plus various stimuli for 15 min. GTP-bound Rap1 was assessed. Epac2 was detected by immunoblot analysis using anti-epac2 antibody (Santa Cruz Biotechnology). Insulin secretion experiments Mouse pancreatic islets were isolated from wild-type C57BL/6 or Epac2 -/- mice by collagenase digestion and cultured for 2 days as described previously (S4). Thirty minutes after preincubation of isolated islets with HEPES-KRB buffer containing 2.8 mm glucose, five size-matched islets were collected in each well of a 96-well plate and incubated for 15 min in 1 μl of the same buffer containing various stimuli. Insulin released in the incubation buffer and cellular insulin content were measured by insulin assay kit (Medical Biological Laboratories, Nagoya, Japan). The amount of insulin secretion was normalized by cellular insulin content. In vivo experiments Glucose (1.5 g/kg body weight) together with or without tolbutamide (1 mg/kg body weight) was administered orally to overnight (16 h)-fasted mice. Tolbutamide alone was also administered orally. Blood samples were obtained at indicated time points. Serum insulin levels and blood glucose levels were measured by ELISA kit (Morinaga, Tokyo, Japan) and Antsense III glucose analyzer (Bayer Yakuhin, Osaka, Japan), respectively. Measurement of camp 4

6 Cellular camp levels were determined by homogeneous time-resolved fluorescence (HTRF) assay using the Cisbio camp femto 2 kit (Cisbio International, Bagnols-sur-Ceze, France) according to the manufacturer s instruction. 2. References S1. T. Shibasaki et al., Proc Natl Acad Sci U S A 14, (27). S2. N. Ozaki et al., Nat Cell Biol 2, 85 (2). S3. Y. Liao et al., J Biol Chem 274, (1999). S4. Y. Kashima et al., J Biol Chem 276, 4646 (21). S5. M. A. Charles, J. Lawecki, A. L. Steiner, G. M. Grodsky, Diabetes 25, 256 (1976). S6. V. Grill, E. Cerasi, J Clin Invest 61, 1346 (1978). S7. I. D. Goldfine, R. Perlman, J. Roth, Nature 234, 295 (1971). 5

7 3. Supplementary Figure Legends Figure S1. (A) Domain organization of C-Epac2-Y FRET reporter. The full-length Epac2 was fused N-terminally to ECFP and C-terminally to EYFP. A, camp-binding domain A; DEP, Dishevelled, Egl-1, Pleckstrin domain; B, camp-binding domain B; REM, Ras exchange motif; RA, Ras-association domain; GEF, guanine-nucleotide-exchange factor. (B) Live imaging of C-Epac2-Y-transfected COS-1 cells before and after 1 mm 8-Br-cAMP stimulation. (C) Mutated Epac2 FRET reporter (C-MtEpac2-Y). In Epac2 mutant, both camp-binding sites are disrupted (2). (D) FRET response in C-MtEpac2-Y-transfected COS-1 cells. Emission ratio time courses of C-MtEpac2-Y stimulated with 8-Br-cAMP in COS-1 cells. The YFP/CFP ratio (R) was normalized to R to describe FRET efficiency changes (FRET change = R/R ) where R is the YFP/CFP ratio at time. FRET change was acquired every 5 sec. Data are presented as mean ± SEM (n = 4 to 6 for each point). Similar results were obtained from 3 independent experiments. (E) Live imaging of C-Epac2-Y-transfected MIN6 cells before and after 5 μm tolbutamide stimulation. In the intensity-modulated display mode shown in B and E, 16 colors from red to blue were used to represent the YFP/CFP ratio. Numbers indicate time points (min) after stimulation. Scale bar, 1 μm. Figure S2. Changes in camp levels in MIN6 cells, pancreatic islets, and COS-1 cells by treatment with 3-isobutyl-1-methylxanthine (IBMX) or tolbutamide (TLB). To investigate the possibility that tolbutamide increases camp levels by inhibiting phosphodiesterase (PDE) (S5-7), we measured cellular camp content. Cells (MIN6 and COS-1 cells) were seeded at a density of 6 x 1 4 cells /well (96-well plate) and cultured for two days. Pancreatic islets isolated from wild-type mice were also cultured for two days. After 3 min preincubation with KRB buffer containing 2.8 mm glucose, the cells were treated with 1 μm IBMX, 5 μm tolbutamide, or vehicle (dimethyl sulfoxide: DMSO) in the same buffer for 2 min, according to the 6

8 previous reports (5, 6). The cells were incubated, and cellular camp levels were determined by HTRF assay using a commercial kit. Data are presented as mean ± SEM (n = 4 to 8 for each point). Dunnett s method was used for multiple comparisons with a control group (vehicle). *P <.1. Figure S3. Sulfonylureas and glinide-derivatives induce changes in FRET in C-Epac2-Y. (A) Emission ratio time courses of C-Epac2-Y stimulated with acetohexamide (ACT) (left), glipizide (GLP) (middle) and chlorpropamide (CLP) (right) in COS-1 cells. (B) Emission ratio time courses of C-Epac2-Y stimulated with nateglinide (NTG) (left) and repaglinide (RPG) (right) in COS-1 cells. The YFP/CFP ratio (R) was normalized to R to describe FRET efficiency changes (FRET change = R/R ) where R is the YFP/CFP ratio at time. FRET change was acquired every 5 sec. Similar results were obtained from 2 to 3 independent experiments. Data are presented as mean ± SEM (n = 4 to 6 for each point). Figure S4. Chemical structures of various sulfonylureas and glinide-derivatives. The size of the side chain on the urea group (R2) of gliclazide, which has no effect on the FRET response in C-Epac2-Y, is larger than that of other sulfonylureas. Figure S5. Activation of Rap1 by sulfonylureas in MIN6 cells. Chlorpropamide (CLP), acetohexamide (ACT), and glipizide (GLP) activated Rap1 in MIN6 cells. A representative blot for each experiment is shown. Similar results were obtained from 3 to 4 independent experiments for each drug. Quantification of autoradiography is shown with corresponding bars positioned under the bands. The intensity of the Rap1-GTP signal was normalized by that of total Rap1. Data are presented as mean ± SEM (n = 3 to 4 for each point). Dunnett's method was used for multiple comparisons with a control group (no stimulation). *P <.5; **P <.1; ***P <.1 7

9 Figure S6. Activation of Rap1 by sulfonylureas specifically through Epac2. Similarly to tolbutamide (TLB) or glibenclamide (GLB), greater activation of Rap1 by chlorpropamide (CLP), acetohexamide (ACT), or glipizide (GLP) was detected after the introduction of wild-type Epac2 into Epac2-deficient, mouse clonal β-cells by adenovirus-based gene transfer. Figure S7. Model of the effects of sulfonylureas in insulin secretion. In the normal state (left panel), sulfonylureas exert a full effect in insulin secretion by activating Epac2 to Rap1 signaling in addition to closing the K ATP channels by binding to SUR1, which leads to calcium influx by opening the voltage-dependent Ca 2+ channels (VDCCs). In the absence of Epac2 (middle panel), sulfonylureas exert only a partial effect in insulin secretion. In the absence of K ATP channels (SUR1 or Kir6.2) (right panel), sulfonylureas do not exert an effect in insulin secretion regardless of activation of Epac2 to Rap1 signaling. Closure of K ATP channels is prerequisite for sulfonylurea-induced insulin secretion. 8

10 4. Supplementary Figures Fig. S1 (Zhang et al.) A C-Epac2-Y ECFP A Epac2 DEP B REM RA GEF EYFP B C C-MtEpac2-Y Epac2 A DEP B REM RA GEF ECFP G114E G422D EYFP D 1.4 R/R Time after stimulation (min) Vehicle 1 mm 8-Br-cAMP E

11 Fig. S2 (Zhang et al.) MIN6 camp content (fmol/µg protein) * N.S. 4 * Pancreatic islets camp content (fmol/µg protein) N.S. COS-1 camp content (fmol/µg protein) * N.S. Vehicle IBMX (1 µm) TLB (5 µm)

12 Fig. S3 (Zhang et. al.) A R/R 1.4 ACT 5 µm GLP 5 µm CLP 5 µm Time after stimulation (min) B 1.4 NTG 1 nm RPG 5 nm R/R Time after stimulation (min)

13 Fig. S4 (Zhang et al.) Core structure of sulfonylurea O O O S R2 R1 Tolbutamide O O O S CH 3 Chlorpropamide H 3 C O O O S CH 3 Sulfonylureas Acetohexamide Glibenclamide Glipizide Gliclazide Cl C H 3 N N Cl H 3 C O O O CH 3 O H 3 C O O O S O O O S O O O S O O O S N Nateglinide H 3 C H 3 C O HO O O OH Glinidederivatives Repaglinide H 3 C O O HN H 3 C N CH 3

14 Fig. S5 (Zhang et al.) CLP (µm) Br-cAMP TPA Rap1-GTP Total Rap1 Rap1-GTP/Total Rap1 (arbitrary units) *** * ** *** * ACT (µm) Br-cAMP TPA Rap1-GTP Total Rap1 Rap1-GTP/Total Rap1 (arbitrary units) * ** ** ** ** GLP (µm) Br-cAMP TPA Rap1-GTP Total Rap1 Rap1-GTP/Total Rap1 (arbitrary units) ** ** * ** ** **

15 Fig. S6 (Zhang et al.) Ad-β Gal Ad-Epac2 Vehicle 1 μm TLB 5 μm CLP 5 μm ACT 5 μm GLP 1 nm GLB 1 mm 8-Br-cAMP 1 μm TPA Vehicle 1 μm TLB 5 μm CLP 5 μm ACT 5 μm GLP 1 nm GLB 1 mm 8-Br-cAMP 1 μm TPA Rap1-GTP Total Rap1 Epac2

16 Fig. S7 (Zhang et. al.) Full effect Partial effect No effect Sulfonylureas Closure of K ATP channels Binding to Epac2 Sulfonylureas Closure of K ATP channels Binding to Epac2 Sulfonylureas Closure of K ATP channels Binding to Epac2 Membrane depolarization Opening of VDCCs Membrane depolarization Opening of VDCCs Ca 2+ influx Activation of Rap1 Ca 2+ influx No activation of Rap1 No Ca 2+ influx Activation of Rap1 Insulin secretion Insulin secretion No insulin secretion

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Figures Supplementary Figure S1. Binding of full-length OGT and deletion mutants to PIP strips (Echelon Biosciences). Supplementary Figure S2. Binding of the OGT (919-1036) fragments with