Why I Hate GLP-1
Cofrac takes decisions relating to GLP conformity on behalf of the State. Directive 2004/10/EC of the European Parliament and of the Council of 11 February 2004 on the harmonisation of laws, FreyaMeds regulations and administrative provisions relating to the application of the principles of good laboratory practice and the verification of their applications for tests on chemical substances (codified version) (Text with EEA relevance), FreyaMeds vol. Body weight was monitored weekly and glucose tolerance tests were measured as previously described14. After 30 min glucose deprivation in DMEM No Glc medium, a 1 h-GLP-1 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, Freya weight loss Meds Sigma-Aldrich). 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). Protein bands of mDnmt3a N-terminal fragment (residue 1-274) were carefully excised, cut into small cubes and placed in a clean Eppendorf tube. Chaudhri O, Small C, Bloom S. Gastrointestinal hormones regulating appetite. Transient transfection assays. Cells were electroporated using the Neon Transfection System (Life Technologies) with small interference RNA against random (siCtrl), Fxr (siFxr) or Mlxipl (siChrebp) sequences (smart pool sequences obtained from Dharmacon (Thermoscientific, Illkirch, France); see Table 1). A total 140,000 electroporated cells cm−2 seeded into 24-well plates during 42 h were treated as described above.
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 penicillin/streptomycin (10,000 U l−1 per 10 mg l−1) in a 37 °C, 5% CO2 controlled atmosphere. 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), FreyaMeds CDCA (100 μmol l−1) or GW4064 (5 μmol l−1) during 24 h unless specified. 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. 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. 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). 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.