glass electrode geology instrument n a p 1000 next generation micropipette puller sutter instrument n a microdrill rwd life science  (Sutter Instrument Company)

Journal: bioRxiv

Article Title: Glucose is a ligand-like trigger for secretion of postprandial GLP-1

doi: 10.64898/2026.01.04.697549

Figure Lengend Snippet: (A and B) K ATP activity is modulated by glucose binding of GK in controlling the secretion of GLP-1. GK -knockdown ( GK -KD) STC-1 cells were reintroduced with glucose-binding-defective GK-G80A (B) or glucose-constitutive-binding GK-L309P (A). Cells were then treated for 1 h with K ATP inhibitors glibenclamide and repaglinide (B) or K ATP openers diazoxide and NN414 (A), at a concentration of 10 µM. After the treatment, GLP-1 secretion was measured following an additional 1-h incubation with different concentrations of glucose. Results are shown as mean ± SEM; n = 8 biological replicates for each treatment, and p value by one-way (B) or two-way (A) ANOVA, followed by Tukey. (C) Schematic illustrating K ATP channels are closed on binding to glucose-bound GK in the stimulation of GLP-1 secretion by high glucose. (D-G) GK in glucose-bound state inhibits K ATP . GK -KD STC-1 (D and E), GK -KD GLUTag cells (D and E), or the volume-regulated anion channels (VRAC)-deficient/ LRRC8A -KO HEK293T cells pre-transfected with Kir6.2-mCherry and Myc-SUR1 (F and G), were infected with lentivirus carrying GFP-tagged GK-G80A or GK-L309P, and then starved for glucose for 2 h. The plasma membrane of the individual cells was then suctioned using a glass micropipette (containing buffer with 2 mM ATP and 200 μM ADP), forming a gigaohm seal (illustrated in the upper panels of D and E). The membrane was then ruptured to achieve whole-cell access, followed by recording the whole-cell K + current evoked by a linear voltage ramp from −140 mV to +60 mV (D and E) or from −120 mV to +120 mV (F and G) over a duration of 400 msec, applied repeatedly every 2 sec. Glucose were either supplemented in the bath solution (extracellular), which was varied from 1 mM (depicted as in grey bar) to 25 mM (green bar), and then returned to 1 mM (washout with 1 mM solution; yellow bar), followed by treatment with 10 µM glibenclamide (blue bar) using a perfusion system during the recording (D and F), or maintained in the pipette solution (cytosolic face) at a constant concentration during the recording, with an 5-min equilibration period after achieving whole-cell access (E and G). The representative trace (lower panel of D, and left panel of F) and the statistical analysis (lower panel of E, G, and right panel of F) of the ramp-induced currents recorded at −90 mV (D and E) or −120 mV (F and G) are shown (mean ± SEM, n = 7 (STC-1 + GK-WT of E), 6 (E, others), 60 (GK-G80A of F), 59 (F, others), 12 (GK-WT of G), or 8 (G, others) cells for each genotype/treatment, with the p value determined by two-way ANOVA, followed by Tukey). In the middle panel of F, the representative steady-state current-voltage (I-V) curves, measured under conditions where current density (cell capacitance) varied by less than 1 pA/pF at each glucose concentration (indicated by triangles in the upper middle panel as an example), are shown. (H) GK in glucose-bound state inhibits K ATP in vitro. HEK293T cells were transfected with Kir6.2 and SUR1 subunits of K ATP , followed by excising a membrane patch and exposing it in the bath solution (intracellular side) containing 2 mM ATP, 200 μM ADP, varying concentrations of glucose, along with bacterially expressed and purified wildtype GK, GK-G80A, and GK-L309P, Steady-state current was continuously recorded at a constant voltage of +60 mV (illustrated in the left panel). The channel open probability was quantified as the percentage of the total opening time within a 1-min analysis window. Data are shown as mean ± SEM; n = 17 (WT, 25 mM glucose), 15 (G80A) or 16 (others) membrane patches for each condition, with the p value determined by two-way ANOVA, followed by Sidak. See also representative 20-sec current traces for wildtype GK in , with downward deflections indicating the channel openings. Experiments in this figure were performed three times.

Article Snippet: Borosilicate glass electrodes (micropipettes) with a tip resistance of 2-4 MΩ were pulled using a Next Generation Micropipette Puller (P-1000; Sutter Instrument) and filled with pipette (cytosolic face) buffer containing 150 mM KCl, 2 mM MgCl 2 , 20 mM HEPES supplemented with 2 mM Mg-ATP, 200 μM ADP (freshly prepared), and the desired concentrations of glucose (supplemented with NMDG and MSA to bring the pH to ∼ 7.2 and final osmolarity to ∼ 300 mOsm).

Techniques: Activity Assay, Binding Assay, Knockdown, Concentration Assay, Incubation, Transfection, Infection, Clinical Proteomics, Membrane, Transferring, In Vitro, Purification