Eight Things To Demystify GLP-1

The OECD Principles of GLP was developed specifically for the conduct of studies which produce data for regulatory decisions. The experiment on GLU-VENUS mice was approved by local ethics committees and conformed to United Kingdom Home Office regulations, the experiment on Tgr5−/− mice were approved by the local animal experimentation committee of the Canton de Vaud (license no. 2614) and the experiment on GF/CONV-R mice was performed with protocols approved by the University of Gothenburg Animal Studies Committee. After 30 min glucose deprivation in DMEM No Glc medium, a 1 h-ColonBroom GLP-1 formula secretion test in response to DMEM without glucose, to DMEM with glucose (5.5 mmol l−1) or in response to DMEM with glucose (5.6 mmol l−1) plus phloretin (0.5 mmol l−1) was performed at 37 °C in DMEM plus 1% DPP-4 inhibitor (Ile-Pro-Ile, Sigma-Aldrich). After permeabilization during 10 min (Tris/NaCl/0.1% TRITON X100), unspecific protein binding sites were masked by Dako Protein Block (DAKO) during 2 h at room temperature. The remaining tissue was washed in cold PBS and frozen at −80 °C in NaOH 2 N. Protein content was assessed according to BCA’s method (Thermoscientific). Cells were lysed in NaOH (0.8 mol l−1) under agitation and total protein content was determined using the BCA Protein Assay Kit (Pierce).



To estimate mitochondrial quantity, 24 h DMSO- and GW4064-treated GLUTag cells were washed with PBS, trypsinized and incubated at 37 °C for 20 min with 100 nmol l−1 MitoTracker Green FM (Molecular Probes). Pieces were washed 5 × in cold HBSS plus 2% horse serum and then incubated for 10 min at 4 °C in HBSS containing 2% horse serum and 1,4-dithiothreitol (DTT, 1 mmol l−1). After additional washing in cold HBSS plus 2% horse serum, ileum pieces were distributed in 48-well plates and stabilized for 3 h at 37 °C in Iscove’s Modified Dulbecco Medium (Life Technologies) containing 10% fetal calf serum (Life Technologies). Peyer’s patches were removed and the ileum was opened longitudinally and cut into 5-mm-long pieces. Ileum (corresponding to the five terminal centimetres of the small intestine) and colon were washed once with phosphate-buffered saline (PBS), opened longitudinally on ice and the intestinal mucosa was scrapped and snap-frozen in liquid nitrogen. As recognized earlier, this can be one of the challenges that small hotels face in their regenerative journey, but with the right resources and support, it can be achieved.



Adults with prediabetes or type 2 diabetes can only be prescribed Wegovy and Saxenda within specialist weight management services. GLP-1 medications like Wegovy may help women manage polycystic ovary syndrome (PCOS), a hormonal condition linked to symptoms such as acne, excessive hair growth, weight gain and fertility issues. ATP measurement (Cell Titre Glow, Promega) on GLUTag cells in response to glucose (5.6 mmol l−1) was performed in the same condition as GLP-1 secretion assays according to the manufacturer’s protocol and luciferase activity was measured using Viktor apparatus (PerkinElmer). In some experiments, cells were deprived of glucose by a 12 h incubation in glucose-free medium (DMEM GLUTamax, Cat. After treatment, GLUTag cells were starved for 30 min in glucose-free Krebs/phosphate buffer (NaCl (120 mmol l−1), KCl (5 mmol l−1), MgCl2 (0.25 mmol l−1), CaCl2 (0.5 mmol l−1) and NaHCO3 (2.2 mmol l−1), pH 7.2) supplemented with diprotin A (100 μmol l−1) and 0.2% BSA. GlutaMAX-1 medium (Cat. No. 21885-025, Invitrogen) with glucose (5.6 mmol l−1), sodium pyruvate (1 mmol l−1) and supplemented with 0.2% BSA containing TβMCA (100 μmol l−1), CDCA (100 μmol l−1) or GW4064 (5 μmol l−1) during 24 h unless specified. The cell supernatants were then transferred into ice-cold microtubes containing an equal volume of diprotin A (100 μmol l−1) in Krebs buffer and centrifuged at 1,500g, 4 °C for 5 min.



Cells were incubated in DMEM containing 0.1% bovine serum albumin (BSA) at 37˚C for two days, and the medium was replaced with medium containing indicated concentrations of metformin. Other chemicals included adenosine 5′-diphosphate sodium salt (ADP), fatty acid-free bovine serum albumin (BSA), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), oligomycin, antimycin A, rotenone, and succinic acid. The last 8 cm of the small intestine, corresponding to the ileum, was placed in cold Hank’s Balance Salt Solution (HBSS, Lonza) containing 2% horse serum (Life Technologies). GlutaMAX-1 medium (Cat. No. 21885-025, Invitrogen) containing glucose (5.6 mmol l−1), sodium pyruvate (1 mmol l−1) and supplemented with 10% FBS. GlutaMAX-1 (catalogue (Cat.) No. 61870-010, Life Technologies) containing glucose (11 mmol l−1), sodium pyruvate (1 mmol l−1) and supplemented with 10% FBS (Life Technologies) and ColonBroom GLP-1 formula penicillin/streptomycin (10,000 U l−1 per 10 mg l−1) in a 37 °C, 5% CO2 controlled atmosphere. No. 11966-025), supplemented with 1% glutamine, sodium pyruvate (1 mmol l−1), 0.2% BSA and sodium lactate (10 mmol l−1) before FXR activation in either lactate (10 mmol l−1)-, glucose (5.6 mmol l−1)- or 2-deoxyglucose (5.6 mmol l−1)-containing medium. GLP-1 content in culture medium was measured as described below.