GLP-1(7–36)NH2, exendin-4, and exendin(9–39) were from Bachem, and custom peptides were from Insight Biotechnology. TMR and FITC conjugates of exendin-4 and/or exendin-phe1 and exendin(9–39) were coupled via K12, as previously described [22 (link)]. BETP was from Sigma-Aldrich.
Exendin 9 39
Manufactured by Bachem
Sourced in Switzerland, Germany
Exendin(9–39) is a peptide used in research applications. It is a truncated version of the exendin-4 peptide and functions as a glucagon-like peptide-1 receptor (GLP-1R) antagonist.
Automatically generated - may contain errors
Lab products found in correlation
Exendin 4, by Bachem (3 mentions)
Glp 1 7 36 nh2, by Bachem (2 mentions)
Ar c 118925xx, by Bio-Techne (1 mentions)
Liraglutide, by Bachem (1 mentions)
Oxyntomodulin, by Bachem (1 mentions)
Glucagon, by Bachem (1 mentions)
Glp 1, by Bachem (1 mentions)
Sitagliptin, by Santa Cruz Biotechnology (1 mentions)
V plex plus proinflammatory panel 1 mouse kit, by Mesoscale (1 mentions)
Mouse rat insulin kit, by Mesoscale (1 mentions)
Actrapid hm penfill, by Novo Nordisk (1 mentions)
Freestyle, by Abbott (1 mentions)
Isradipine, by Alomone (1 mentions)
L 168 049, by Bio-Techne (1 mentions)
ω agatoxin, by Alomone (1 mentions)
Pertussis toxin ptx, by Merck Group (1 mentions)
Cck 8, by Merck Group (1 mentions)
L 371 257, by Bio-Techne (1 mentions)
Ex9 39, by Bachem (1 mentions)
80 insmsu e01, by ALPCO (1 mentions)
13 protocols using exendin 9 39
1
Fluorescent Exendin Peptides Synthesis
2
Synthetic Peptides for Functional Assays
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Sequences of the synthetic peptides used in the functional experiments are shown in Fig 1 . Synthetic hGLP-1(7–36)amide (referred throughout as hGLP-1), synthetic human glucagon, exendin-4 and exendin(9–39) were purchased from Bachem (Torrence, CA). zfGLP-1, zebrafish glucagon and zfGLP-2 were synthesized by the Rockefeller University Proteomics Facility. Zebrafish PACAP-38amide was synthesized by the Protein and Carbohydrate structure facility at the University of Michigan. The homogeneity of all peptides used in the functional experiments was checked by HPLC and mass spectroscopy and was >99%.
3
Electrophysiological and Imaging Techniques
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Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich. CNO, Ro51 and AR-C 118925XX were from Tocris (Biotechne), Exendin 9-39 was from Bachem. A Gq-DREADD expression plasmid was purchased from Addgene. Drugs for imaging and electrophysiology experiments were applied directly onto cells using a custom-made gravity-fed perfusion system. To reduce flow-induced artefacts, a constant flow of external solution was applied onto cells during baseline recordings and switched to a drug solution during drug application. Unless otherwise stated, recordings were performed at room temperature (20–24 °C).
Standard saline (138 buffer) contained (in mM): NaCl (138), KCl (4.5), HEPES (10.0), NaHCO3 (4.2), NaH2PO4 (1.2), CaCl2 (2.6), MgCl2 (1.2); pH 7.4 with NaOH. The following concentrations of D-glucose were used: for ATP secretion experiments 0.1 mM, for electrophysiological experiments 1 mM, and for Fura-2 imaging experiments, 5 mM glucose. The Ringer’s solution used in Ussing chamber experiments contained (in mM): NaCl (120), KCl (3.0), MgCl2 (0.5), CaCl2 (1.25), NaHCO3 (23.0), and D-glucose (10.0), and constantly bubbled with carbogen (95% O2/5% CO2), pH 7.4 ± 0.2.
Standard saline (138 buffer) contained (in mM): NaCl (138), KCl (4.5), HEPES (10.0), NaHCO3 (4.2), NaH2PO4 (1.2), CaCl2 (2.6), MgCl2 (1.2); pH 7.4 with NaOH. The following concentrations of D-glucose were used: for ATP secretion experiments 0.1 mM, for electrophysiological experiments 1 mM, and for Fura-2 imaging experiments, 5 mM glucose. The Ringer’s solution used in Ussing chamber experiments contained (in mM): NaCl (120), KCl (3.0), MgCl2 (0.5), CaCl2 (1.25), NaHCO3 (23.0), and D-glucose (10.0), and constantly bubbled with carbogen (95% O2/5% CO2), pH 7.4 ± 0.2.
4
Dietary and Pharmacological Interventions in Mice
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Mice were fed either chow (5021 LabDiet) or WSD (ENVIGO) for 8 weeks. The diets were purchased from. Diet compositions are presented in online supplemental table 1 ).
For exendin-4 delivery, Alzet osmotic mini pumps containing either 0.9% NaCl as a vehicle or exendin-4 (2 nmol/kg/day; HY-1344, MedChemTronica) were used for a period of 6 weeks. The mini pumps were primed for 24 hours before use and were implanted subcutaneously in the dorsal area under anaesthesia.
For exendin 9-39 administration (4017799.0500, Bachem), a dose of 5 µg/kg of body weight was injected intraperitoneally using 0.9% NaCl 20 min before performing the OGTT.
HDCA (H3878, sigma) was resuspended in olive oil and administered by intragastric gavage 3 days a week at the dose of 50 mg/kg of body weight.
For exendin-4 delivery, Alzet osmotic mini pumps containing either 0.9% NaCl as a vehicle or exendin-4 (2 nmol/kg/day; HY-1344, MedChemTronica) were used for a period of 6 weeks. The mini pumps were primed for 24 hours before use and were implanted subcutaneously in the dorsal area under anaesthesia.
For exendin 9-39 administration (4017799.0500, Bachem), a dose of 5 µg/kg of body weight was injected intraperitoneally using 0.9% NaCl 20 min before performing the OGTT.
HDCA (H3878, sigma) was resuspended in olive oil and administered by intragastric gavage 3 days a week at the dose of 50 mg/kg of body weight.
5
Peptide Comparison for GLP-1 Receptor
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Exendin-4, exendin(9-39), and GLP-1 (7–36)NH2 were from Bachem; Liraglutide, Lixisenatide, and Dulaglutide from Imperial College Healthcare NHS Trust pharmacy; and custom peptides from Insight Biotechnology.
6
Synthesis and Purification of OX-SR and OX-SR-Glu3
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OX-SR and OX-SR-Glu3 were synthesized by Insight Biotechnology Ltd. (Middlesex, UK) using solid phase peptide synthesis (SPPS) methodology and purified by reverse-phase preparative HPLC. Peptide purity was greater than 95%. Throughout, OX-SR and OX-SR-Glu3 were administered in a zinc-based diluent. Oxyntomodulin, GLP-1, glucagon and exendin 9–39 were all purchased from Bachem (Bubendorf, Switzerland). OX-SR-Glu3 has the same peptide structure as the long-acting OXM analogue OX-SR with a substitution of glutamic acid at position 3 to eliminate activity of the peptide at the glucagon receptor [6] (link).
7
Metabolic Phenotyping of Mice on High-Fat Diet
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Metabolic phenotype was assessed by glucose and insulin tolerance tests (GTT/ITT) performed at 1, 4, and 12 weeks of HFD feeding. For a GTT, mice fasted 6 h. After intraperitoneal (IPGTT) or oral (OGTT) injection of glucose (2 g/kg body weight) blood glucose was monitored from the tail vein after 15, 30, 60, 90, and 120 min by using a glucometer (Freestyle, Abbot). For active GLP-1 measurements, 25 mg/kg sitagliptin (Cat#sc-364620, Santa Cruz) was injected i.p. 30 min before oral glucose application. To block GLP-1 or parasympaticus action, 236 µg/kg exendin (9-39) (#H-8740, Bachem) or 5 mg/kg atropine (#A0257-5G, Sigma), respectively, were i.p. injected at timepoint −30 min prior glucose application. ITT was performed after 3 h of fasting by injecting 1–2 U/kg body weight insulin i.p. (Actrapid HM Penfill, Novo Nordisk). Glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after injection.
Plasma insulin, GLP-1, TNF, and IL-6 were quantified by electrochemiluminescence (MESO SECTOR S 600) by using kits from Meso Scale Diagnostics (MSD, Rockville, MD, USA), according to the manufacturer’s instructions: Mouse/Rat Insulin Kit (#K152BZC), V-PLEX Plus Proinflammatory Panel 1 Mouse Kit (#K15048G), V-PLEX GLP-1 Active Kit vers. 2 (#K15030D).
Plasma insulin, GLP-1, TNF, and IL-6 were quantified by electrochemiluminescence (MESO SECTOR S 600) by using kits from Meso Scale Diagnostics (MSD, Rockville, MD, USA), according to the manufacturer’s instructions: Mouse/Rat Insulin Kit (#K152BZC), V-PLEX Plus Proinflammatory Panel 1 Mouse Kit (#K15048G), V-PLEX GLP-1 Active Kit vers. 2 (#K15030D).
8
Investigating Glucagon-like Peptide Signaling
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GLP-1(7–36), GLP-1(9–36), exendin-4 and exendin(9–39) were from Bachem (Weil am Rhein, Germany), the glucagon receptor (GCGR) antagonist REMD2.59 was from REMD Bioptherapeutics (Camarillo, CA, USA) and pertussis toxin (PTX) was from Sigma (Gillingham, Dorset, UK). The Ca2+ channel blockers ω-agatoxin and isradipine, respectively blocking voltage-gated Ca2+ channels sensitive to ω-agatoxin (P/Q-type Ca2+ channels) and L-type Ca2+ channels, were from Alomone Labs (Jerusalem, Israel). The glucagon receptor antagonists (GRA) L-168049 and REMD2.59 were from Tocris Bioscience (Bristol, UK) and REMD Biotherapeutics Inc (Camarillo, CA, USA), respectively. Sitagliptin was obtained from Stratech Scientific (Ely, UK). The protein kinase A (PKA) inhibitor 8-Br-Rp-cAMPS was purchased from BioLog Life Science Institute (Bremen, Germany).
9
Pharmacological Modulation of Feeding Behavior
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For GLP-1R agonism, the SPF mice, and the ABX mice received an intraperitoneal injection of 4.2 μg/kg exenatide (Ex-4; Bachem)107 (link) dissolved in 0.9% saline 30 minutes before behavior test. For GLP-1R antagonism, the ABX mice, the SPF mice, and the ABX-SDV mice were intraperitoneally injected with the 500 μg/kg exendin (9-39) (Ex(9-39); Bachem)108 (link) dissolved in 0.9% saline 30 min before behavior test.
For CCK-induced food intake suppression, the SPF mice were intraperitoneally delivered with 8 μg/kg CCK-8 (Sigma-Aldrich) dissolved in 0.9% saline 30 min before behavior test.
For oxytocin receptor antagonism, 300 µg/kg L-371,257 (Tocris Bioscience) was administrated to mice intranasally. The L-371,257 powder was dissolved in 10% dimethyl sulfoxide (DMSO) in phosphate buffered saline (PBS). The solution was administered two microliters into each nostril 20 min before the behavior test.
For CCK-induced food intake suppression, the SPF mice were intraperitoneally delivered with 8 μg/kg CCK-8 (Sigma-Aldrich) dissolved in 0.9% saline 30 min before behavior test.
For oxytocin receptor antagonism, 300 µg/kg L-371,257 (Tocris Bioscience) was administrated to mice intranasally. The L-371,257 powder was dissolved in 10% dimethyl sulfoxide (DMSO) in phosphate buffered saline (PBS). The solution was administered two microliters into each nostril 20 min before the behavior test.
10
Pancreatic Islet Isolation and Perifusion
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The pancreas was inflated through the pancreatic duct with collagenase (7.5 mg/mL, Sigma C7657) in HBSS (5 mM glucose, 1 mM MgCl2), excised and incubated at 37°C for 12 minutes. The digestion was quenched with ice-cold HBSS (5 mM glucose, 1 mM MgCl2, 1 mM CaCl2). The digested pancreas was washed with HBSS, and islets were separated using a Histopaque gradient (Sigma, 10771). Islets were handpicked to achieve final purity of 95-99% and incubated in complete RPMI (10% FBS, 1% penicillin/streptomycin) overnight at 37°C and 5% O2 before perifusion.
A Biorep perifusion system (Biorep Technologies) was used to stimulate islet hormone secretion in vitro. Solutions were prepared in KRBH buffer (115 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 24 mM NaHCO3, 10 mM HEPES, and 1% BSA; pH = 7.4). Islets were equilibrated for 48 minutes with 2.8 mM glucose and then perifused in intervals in the indicated experimental conditions. Flow rate (100 μl/min) and temperature (37°C) remained constant, and the type of treatment is indicated at the top of each figure (Arginine, Sigma, #A5006; GLP-1 7-36, Bachem, 4030663; GIP-(D-Ala2 (link))-GIP, Bachem #4054476; Exendin 9-39, Bachem #4017799). Effluent fractions collected at 2-minute intervals were stored at -80°C for further assays. Insulin concentration was measured using a Mouse Ultrasensitive or High Range Insulin ELISA (ALPCO, 80-INSMSU-E01 or 80-INSMSH).
A Biorep perifusion system (Biorep Technologies) was used to stimulate islet hormone secretion in vitro. Solutions were prepared in KRBH buffer (115 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 24 mM NaHCO3, 10 mM HEPES, and 1% BSA; pH = 7.4). Islets were equilibrated for 48 minutes with 2.8 mM glucose and then perifused in intervals in the indicated experimental conditions. Flow rate (100 μl/min) and temperature (37°C) remained constant, and the type of treatment is indicated at the top of each figure (Arginine, Sigma, #A5006; GLP-1 7-36, Bachem, 4030663; GIP-(D-Ala2 (link))-GIP, Bachem #4054476; Exendin 9-39, Bachem #4017799). Effluent fractions collected at 2-minute intervals were stored at -80°C for further assays. Insulin concentration was measured using a Mouse Ultrasensitive or High Range Insulin ELISA (ALPCO, 80-INSMSU-E01 or 80-INSMSH).
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