In mice and humans, we also measured the gene expression of was barely detectable in mouse – and -cells, whereas it was well expressed in human being -cells. performed having a validated antibody. The effects of dapagliflozin, empagliflozin, and sotagliflozin on glucagon and insulin secretion were assessed using isolated rat, mouse and human being islets and the perfused mouse pancreas. Finally, we tested the long-term effect of SGLT2i on glucagon gene manifestation. Results SGLT2 inhibition in mice improved the plasma glucagon/insulin percentage in the fasted state, an effect correlated with a decrease in glycemia. Gene manifestation analyses and immunodetections showed no SGLT2 mRNA or protein manifestation in rodent and human being islet cells, but moderate SGLT1 mRNA manifestation in human being -cells. However, practical experiments on rat, mouse, and human being (29 donors) PRN694 islets and the perfused mouse pancreas did not identify any direct effect of dapagliflozin, empagliflozin or sotagliflozin on glucagon and insulin secretion. SGLT2i did not impact glucagon gene manifestation in rat and human being islets. Conclusions The data indicate the SGLT2i-induced increase of the plasma glucagon/insulin percentage does not result from a direct action of the gliflozins on islet cells. perfused mouse PRN694 pancreas. 5) We verified the effects of the gliflozins in mice. 6) Finally, we tested the long-term effect of SGLT2i on glucagon gene manifestation. 2.?Methods 2.1. Study approval The experiments were authorized by the committees for animal welfare (2014/UCL/MD/016 and 2018/UCL/MD/18) and human being islets (2017/12JUL/369) in the Universit Catholique de Louvain and adopted the regulatory conditions of Boehringer Ingelheim’s corporate and business policy in accordance with German legislation. 2.2. Models and cells preparation 2.2.1. Rodent strains and islet preparation Wistar-Han rats and C57BL/6N mice (6C12 weeks) were utilized for all experiments, except for gene manifestation, which was carried out using Glu-Venus [29] and RIPYY mice [30]. Islets were isolated by collagenase and cultured over night in RPMI 1640 medium comprising 11?mM (rat) or 7?mM (mouse) glucose and 10% FBS. 2.2.2. Human being islets The origin and characteristics of the human being islet preparations are outlined in Supplementary Table?S1. After shipment, the islets were cultured for 2C17 days (mean: 5.5?d; median: 5?d) in RPMI 1640 medium containing 5?mM glucose and 10% FBS or PIM medium (Prodo Labs). 2.3. Fluorescence-activated cell sorting and gene manifestation measurements 2.3.1. FACS Dispersed islet cells were FACS-sorted using methods adapted to the different species (Supplementary Number?S1). 2.3.2. cDNA preparation RNA was extracted using Dynabead-oligo dT or TriPure and reverse transcribed into cDNA. 2.3.3. qPCR TaqMan probes and SYBR Green were used. See Supplementary Table?S2 for probe units and primers. Changes in gene mRNA levels normalized to the people of research genes (experiments Medicines (dapagliflozin, PRN694 empagliflozin, and sotagliflozin, 1C10?mg/kg BW) or vehicle (DMSO) were administered by oral gavage to mice either once or one dose during 3 consecutive days. ELISA kits were used to assay plasma glucagon (Mercodia) and insulin (Crystal Chem). 2.8. Secretion experiments 2.8.1. Incubation experiments These experiments were performed with rat and human being islets (10 islets/100?L medium). The medium contained (in mM): 137 NaCl, 5.4 KCl, 1.3 CaCl2, 0.81 MgSO4, 0.34 NaH2PO4, 0.44 KH2HPO4?+?1?mg/mL BSA, and was at pH 7.4. Islets were managed for 30?min inside a medium containing 25 (rat) or 11.1?mM (human being) glucose before being KIP1 transferred inside a medium containing 1 mM glucose and the respective treatments. One hour later on, glucagon was identified using a Fluorescent EIA Kit (Phoenix Pharmaceuticals). 2.8.2. Dynamic secretion experiments Experiments on perifused mouse and human being islets and perfused mouse pancreas were PRN694 performed as previously explained [35]. The medium contained (in mM): 124 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgCl2, 20 NaHCO3?+?1?mg/mL BSA, and was at pH 7.4. Except where otherwise indicated, it was supplemented having a 6?mM combination (for the perifused islets) or 2?mM combination (for the perfused pancreas) of amino acids (see number legends). Insulin (home-made assay) and glucagon (Merck Millipore) were measured by radioimmunoassays. 2.9. Statistical methods Statistical significance of variations between means was evaluated by combined t-tests or one-way ANOVA followed by Tukey’s or Fisher’s LSD test as explained in the number legends and results. 3.?Results 3.1. experiments To verify the effectiveness of the gliflozins used, dapagliflozin, empagliflozin, sotagliflozin (1?mg/kg BW), or vehicle (DMSO) were administered by oral gavage in mice taken care of for 16?h in metabolic cages and their urine was collected (Number?1A). Glycemia tended to decrease during this 16-h period but gliflozins did not exacerbate this drop (Number?1BCC). As expected, all the gliflozins strongly improved glucosuria,.