In cohort 1 and 2, the intestine sample was collected immediately after mouse sacrifice, quickly cleaned, frozen in liquid nitrogen and then stored in -80°C. Collection was done in both overnight fasted mice and freely fed mice. Total RNA was prepared from the tissues with TRIzol reagent (Cat. T9424, Sigma, St. Louis, MO). TaqMan RT-PCR reaction was used to quantify GIP mRNA using the 7900 HT Fast real-time PCR System (Applied Biosystems, Foster City, CA) as previously described [31 (link)]. TaqMan primer for mouse GIP (Mm00433601-m1) and 18S (AIQJA2B, P N4331348) were purchased from the Applied Biosystems. The GIP mRNA signal was normalized with ribosome 18S RNA. In the tissue culture, GIP protein in the culture supernatant was determined using an ELISA kit (EZRMGIP-55K, EMD Millipore, Billerica, MA 01821). In cohort 1, GIP mRNA was tested at 10 wks on HFD. In cohort 2, the test was done at 6 weeks after surgery.
Ezrmgip 55k
Manufactured by Merck Group
Sourced in United States, Germany
The EZRMGIP-55K is a laboratory equipment product manufactured by Merck Group. It is a precision-engineered instrument designed for various applications in research and analytical laboratories. The core function of the EZRMGIP-55K is to perform high-efficiency magnetic separation and isolation of target biomolecules or particles.
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21 protocols using ezrmgip 55k
1
Quantifying Intestinal GIP Expression
2
Quantification of Gut Hormones
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Concentrations of insulin, total GIP, and total GLP-1 were measured using ELISA kits for insulin (Cat# AKRIN-011T, FUJIFILM Wako Shibayagi Corporation, Gunma, Japan), total GIP (Cat# EZRMGIP-55K, Merck, Billerica, MA, US), and total GLP-1 (Cat# K1503PD-1, Meso Scale Discovery, Rockville, MD, US), respectively.
3
Quantifying GLP-1 and GIP Levels in Obese Rats
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F344 rats were fed HFD from 5 weeks old in order to develop diet-induced obesity (defined as DIO-F344 rats). DIO-F344 rats (41 weeks old, baseline BW 532 ± 16 g) were randomized based on their BW, and were bled from the tail vein at indicated time points after drug administration (N = 6 per group, total N = 18). Plasma total GLP-1 levels were determined using sandwich ELISA, which had been established by Takeda Pharmaceutical Company Limited [33 (link)] . Plasma total GIP levels were measured using a commercially available total GIP ELISA kit (EZRMGIP-55K, Merck Millipore, Burlington, MA, USA).
4
Metabolic Biomarker Quantification Protocol
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Terminal plasma measurements were completed by the Mouse Metabolic Phenotyping Center (MMPC) at The University of Cincinnati, Cincinnati, Ohio (www.uc.edu/labs/mmpc.html ). Assays include leptin (#EZRL-83K, EMD Millipore, Billerica, MA), gastric inhibitory peptide (GIP) (#EZRMGIP-55K, EMD Millipore,) total GLP-1 (EZGLP1T, EMD Millipore), C-peptide (EZRMCP2-21K, EMD Millipore) and glucagon (#48-GLUHU-E01, Alpco, Salem, NH). In addition, insulin concentrations were determined using an Insulin ELISA (#90060, Crystal Chem INC., IL). All assays were performed according to the manufacturer’s specifications.
5
Metabolic Biomarker Profiling in OGTT
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All biochemical and hormonal parameters were measured in collected plasma samples from the OGTT. The chemical analysis was carried out to determine the concentration of liver enzymes such as cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) using 7189 Clinical Analyzer (HITACHI, Tokyo, Japan). The hormonal parameters, including insulin, GLP-1, and GIP, were estimated using the following enzyme-linked immunoassay (ELISA) kits, respectively: Rat Insulin ELISA Kit (CC-90010, Crystal chem, Elk Grove Village, IL, USA), Multi Species GLP-1 Total ELISA (EZGLP1T-36K, Merck Millipore, Burlington, MA, USA), and Rat/Mouse GIP (total) ELISA (EZRMGIP-55K, Merck Millipore, Burlington, MA, USA). Insulin, GLP-1, and GIP levels were calculated following the manufacturer’s instructions. Each assay was performed in duplicate from the indicated samples.
6
Plasma GLP-1 and Related Metabolite Measurement
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For the measurement of plasma GLP-1, blood samples were collected in ice-cooled BD Microtainer blood collection tubes coated with K2EDTA (36784, BD) followed by addition of 50 µM DPP-IV inhibitor (DPP4, Merck/millipore), while culture medium was collected plus 1% DPP-IV inhibitor and centrifuged for 5 min at 4 °C at 12,000 × g. Active GLP-1 levels were determined by using an active GLP-1 ELISA kit (EGLP-35K, Merck/millipore) according to the manufacturer’s instructions. For the measurement of insulin, a mouse insulin ELISA kit (EZRMI-13K, Merck/millipore) was used. For the measurement of serum levels of total T3, a T3 (total) (Mouse/Rat) ELISA Kit (KA0925, Abnova) was used. For the measurement of GIP, a Rat/Mouse GIP (total) ELISA kit (EZRMGIP-55K, Merck/millipore) was used. For the measurement of cAMP, cAMP was extracted from frozen intestine tissue samples by lysis buffer and analyzed by a mouse cAMP Assay kit (ab138880, Abcam). Intracellular cAMP was extracted from STC-1, NCI-H716 cells and intestinal organoids, and then analyzed according to the manufacturer’s instruction.
7
Pharmacological Evaluation of Gut Hormone Modulators
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PEG200, HCl, Pefabloc, and acetaminophen were purchased from Sigma Aldrich (Cat. No. P3015, 1.00317, 76,307, and A7085; Soborg, Denmark). BIBO3304 (Y1 antagonist), BIIE0246 (Y2 antagonists), and BIBN4096 (CGRP antagonist) were purchased from Tocris (Cat. No. 2412, 1700 and 4561; Bristol, UK). Somatostatin receptor antagonist was purchased from Bachem (iSST, Cat. No. BIM-23627; Bubendorf, Switzerland). Exendin 4 (Ex4) and Exendin (9–39) (Ex(9–39)) were both purchased from Anaspec (Cat. No. ANA24463 and ANA24467; Cambridge Bioscience, Cambridge, UK). Obestatin was kindly provided by Annette Beck-Sickinger and synthesized as previously described [12 (link)]. ELISAs for GLP-1 and insulin were obtained from MesoScale (Cat. No. L4503PA and K152BZC; MesoScale Diagnostics, Maryland, USA). GIP and total ghrelin were acquired from Merck/Millipore (Cat. No. EZRMGIP-55K and EZRGRT-91K; Merck/Millipore Darmstadt, Germany). PYY ELISA was purchased from Crystal Chem (Cat. No. 81501; Zaandam, Netherlands). An acetaminophen kit was obtained from Sekisui Diagnostics (Cat. No. 506–10; Sekisui, Lexington, MA, USA). Cpd1324 was synthesized by InterBioScreen Ltd. as previously described [10 (link)] and formulated with a vehicle containing 3.2 μM (−5.5 M) of zinc ions for all of the in vivo experiments.
8
Oral Glucose Tolerance Test in Rats
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An OGTT was conducted after rats were fed an HFS diet for 6 weeks. Rats were fasted overnight (16-18 hours) and baseline (fasting) blood was collected twice (at -15 minutes and 0 minutes) from the tail vein. Following baseline (0 minutes) blood collection, a glucose solution was orally administered at 2 g/kg containing 0.5% hydroxypropyl methylcellulose (10 mL/kg). Canagliflozin, added to the glucose solution, was administered at 3 or 10 mg/kg. Systemic blood was collected from the tail vein into tubes containing heparin (final concentration, 50 IU/mL; Ajinomoto Company, Inc., Tokyo, Japan), aprotinin [500 kallikrein inhibitor (KI) units/mL; Wako Pure Chemical Industries, Ltd., Osaka, Japan], and the DPP-IV inhibitor (50 μM; DPP4-010; Merck Millipore Co., Billerica, MA) at 0, 15, 30, 60, 90, and 120 minutes after the administration of glucose.
Plasma was separated by centrifugation at 2,300 × g for 10 minites at 4°C and frozen at -80°C until glucose, insulin, GLP-1, and GIP were measured. Plasma glucose, insulin, and total GLP-1 concentrations were measured using Glucose CII test (Wako), rat insulin enzyme-linked immunosorbent assay (ELISA; AKRIN-010T; Shibayagi Co., Ltd., Gunma, Japan), multi-species GLP-1 total ELISA (EZGLP1T-36K; Merck Millipore, Darmstadt, Germany), and rat/mouse GIP (total) ELISA (EZRMGIP-55K, Merck Millipore) kits, respectively.
Plasma was separated by centrifugation at 2,300 × g for 10 minites at 4°C and frozen at -80°C until glucose, insulin, GLP-1, and GIP were measured. Plasma glucose, insulin, and total GLP-1 concentrations were measured using Glucose CII test (Wako), rat insulin enzyme-linked immunosorbent assay (ELISA; AKRIN-010T; Shibayagi Co., Ltd., Gunma, Japan), multi-species GLP-1 total ELISA (EZGLP1T-36K; Merck Millipore, Darmstadt, Germany), and rat/mouse GIP (total) ELISA (EZRMGIP-55K, Merck Millipore) kits, respectively.
9
Oral d-Allulose Effects on Gut Hormones
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The blood samples were collected from the portal vein of male mice fasted overnight (18:00 to next 10:00) under isoflurane anesthesia at 0, 1, 2, and 3 h after p.o. administration of d -allulose (0.3 and 1 g kg−1, 10 ml kg−1) or saline (10 ml kg−1), respectively. The sampling syringe contained heparin (final concentration; 50 IU ml−1), aprotinin (final concentration; 500 kIU ml−1), and DPP-IV inhibitor vildagliptin (final concentration; 10 µM). Plasma was collected after centrifugation (3000 × g, 10 min at 4 °C) and stored at –80 °C until assay. Active GLP-1, total GIP, PYY, and CCK levels were measured using GLP-1 (Active) ELISA (EGLP-35K; Millipore), Rat/Mouse GIP (total) ELISA (EZRMGIP-55K; Millipore), Mouse/Rat PYY EIA (YK081, Yanaihara Institute, Inc.), and Human/Rat/Mouse Cholecystokinin Octapeptide (26-33, non-sulfated) EIA kits (Phoenix Pharmaceuticals, Inc.), respectively.
10
Glucose and Lipid-Induced Incretin Secretion in Mice
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We examined glucose-induced or lipid-induced incretin secretion in mice as described in the Supplemental Methods and in a previous report [10 (link)]. Total GIP and total GLP-1 were assayed by commercial ELISA kits (GIP: EZRMGIP-55K, GLP-1: EZGLP1T-36K; Millipore, Burlington, MA, USA).
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