no insulin or dapagliflozin, #p 0.05 em vs /em . K+ concentration emulating those produced by exogenous insulin but low expression of and was also detected. We correlated Sst release and -cell cytoplasmic Ca2+ ([Ca2+]i) in Sst-Cre-GCaMP3 islets. Three examples of recordings of [Ca2+]i in individual -cells within intact pancreatic islets are shown in Fig. 1a. The glucose responsiveness was variable: spontaneous [Ca2+]i oscillations were observed in 273% of the cells at 1 mM glucose, which increased to 487% (p 0.05 vs 1 mM) at 4 mM and 825% at 20 mM glucose (p 0.001 vs 1 mM; 79 cells in 7 islets from 7 mice). Increasing glucose from 1 to 4 and 20 mM stimulated Sst release by 100% and 1000%, respectively (Fig. 1b), responses that were associated with comparable increases in the frequency of the [Ca2+]i oscillations (Fig. 1c). When applied at 1 mM glucose, the KATP channel blocker tolbutamide (0.2 mM) produced a 5-fold increase in the frequency of the [Ca2+]i oscillations (Fig. 1d and Extended Data 1a). Conversely, the KATP channel activator diazoxide and the L- and R-type Ca2+ channel blockers isradipine and SNX-482, respectively, abolished or reduced glucose-induced [Ca2+]i oscillations in most -cells and strongly inhibited Sst secretion (Fig. 1e-g and Extended Data 1 and ?and2).2). Sst secretion involves intracellular Ca2+ release by a mechanism sensitive to ryanodine and thapsigargin8 (Extended Data 2a). The inhibitory effect of thapsigargin on Sst secretion correlated with an average 40% decrease in the frequency of the [Ca2+]i oscillations (Extended Data 2e). Open in a separate window Physique 1 Regulation of somatostatin secretion by Ca2+.but in the presence of 100 nM insulin and 1 nM dapagliflozin (Dapa). The dotted line shows data for insulin-unresponsive cells. Data in are mean values S.E.M. in of 36 insulin-responsive -cells from 2 mice. but measuring the effect of lowering [K+]o from 4.7 to 2.7 mM. Bar graph in ((which encodes SGLT2) is usually low in mouse -cells and that of (encoding SGLT1) is usually higher (although still lower than transcripts encoding GLUT1-3; see Supplementary Table 1 and 24). The low expression of SGLT1/2 would be in agreement with the small size of the current (~1 pA) in -cells inhibited by high (M) concentrations of dapagliflozin14. In kidney cells, insulin selectively activates SGLT2 (via an effect involving protein phosphorylation) with little effect on SGLT125 but it remains possible that SGLT1 is usually insulin-sensitive in -cells. Dapagliflozin has been reported to stimulate glucagon secretion both in -cells, the possibility that the dapagliflozin-induced suppression of Sst secretion reflects an off-target SGLT2-impartial effect remains possible, comparable to what was recently reported for the related compound canagliflozin28. Ultimately, to conclusively demonstrate that SGLT1 or 2 are functional in -cells, studies would need to be conducted using -cell-specific ablation of and/or measurements of glucagon secretion were performed using the perfused mouse pancreas. Briefly, the aorta was ligated above the coeliac artery and below the superior mesenteric artery and then cannulated. The pancreas was perfused with KRB made up of glucose and CYN154806 at a velocity of 0.24 Ki67 antibody ml/min using an Ismatec Reglo Digital MS2/12 peristaltic pump. The perfusate was maintained at 37C using a Warner Instruments temperature control unit TC-32 4B in conjunction with a tube heater (Warner Instruments P/N 64-0102) and a Harvard Apparatus heated rodent operating table. The effluent was collected in intervals of 1 1 min. Samples were subsequently stored at -80C. Glucagon content in perfusate were measured using U-plex glucagon ELISA (Meso Scale Discovery), according to the manufacturers protocol. Intracellular [Ca2+] measurements [Ca2+]i measurements were performed as described previously35. Islets were imaged in a heated chamber at 37oC placed on an inverted LSM510 confocal microscope (Zeiss; Oberkochen, Germany) using a 40X oil objective (NA1.4). The pinhole diameter was kept constant, and frames of 256×256 pixels were taken every 1-3 s. Parallel measurement of membrane potential and [Ca2+]i The electrophysiological measurements were performed in intact islets essentially using the perforated-patch whole-cell technique in the voltage- or current-clamp modes in -cells. Parallel measurements of [Ca2+]i and membrane potential were performed using an Axioskop 2FS microscope (Zeiss, Oberkochen, Germany) equipped with a 40x/0.8 objective, Lambda DG-4 exciter (Sutter Instruments, USA) and Orca-R2 cooled CCD camera (Hamamatsu, Japan). Images were acquired using an open-source Micromanager software (developed at Ron Vales lab, UCSF, San Francisco, USA) and.Representative of 4 cells (n=3 mice). islets are shown in Fig. 1a. The glucose responsiveness was variable: spontaneous [Ca2+]i oscillations were observed in 273% of the cells at 1 mM glucose, which increased to 487% (p 0.05 vs 1 mM) at 4 mM and 825% at 20 mM glucose (p 0.001 vs 1 mM; 79 cells in 7 islets from 7 mice). Increasing glucose from 1 to 4 and 20 mM stimulated Sst release by 100% and 1000%, respectively (Fig. 1b), responses that were associated with comparable increases in the frequency of the [Ca2+]i oscillations (Fig. 1c). When applied at 1 mM glucose, the KATP channel blocker tolbutamide (0.2 mM) produced a 5-fold increase in the frequency of the [Ca2+]i oscillations (Fig. 1d and Extended Data 1a). Conversely, the KATP channel activator diazoxide and the L- and R-type Ca2+ channel blockers isradipine and SNX-482, respectively, abolished or reduced glucose-induced [Ca2+]i oscillations in most -cells and strongly inhibited Sst secretion (Fig. 1e-g and Extended Data 1 and ?and2).2). Sst secretion involves intracellular Ca2+ release by a mechanism sensitive to ryanodine and thapsigargin8 (Extended Data 2a). The inhibitory effect of Mycophenolic acid thapsigargin on Sst secretion correlated with an average 40% decrease in the frequency of the [Ca2+]i oscillations (Extended Data 2e). Open in a separate window Figure 1 Regulation of somatostatin secretion by Ca2+.but in the presence of 100 nM insulin and 1 nM dapagliflozin (Dapa). The dotted line shows data for insulin-unresponsive cells. Data in are mean values S.E.M. in of 36 insulin-responsive -cells from 2 mice. but measuring the effect of lowering [K+]o from 4.7 to 2.7 mM. Bar graph in ((which encodes SGLT2) is low in mouse -cells and that of (encoding SGLT1) is higher (although still lower than transcripts encoding GLUT1-3; see Supplementary Table 1 and 24). The low expression of SGLT1/2 would be in agreement with the small size of the current (~1 pA) in -cells inhibited by high (M) concentrations of dapagliflozin14. In kidney cells, insulin selectively activates SGLT2 (via an effect involving protein phosphorylation) with little effect on SGLT125 but it remains possible that SGLT1 is insulin-sensitive in -cells. Dapagliflozin has been reported to stimulate glucagon secretion both in -cells, the possibility that the dapagliflozin-induced suppression of Sst secretion reflects an off-target SGLT2-independent effect remains possible, similar to what was recently reported for the related compound canagliflozin28. Ultimately, to conclusively demonstrate that SGLT1 or 2 are functional in -cells, studies would need to be conducted using -cell-specific ablation of and/or measurements of glucagon secretion were performed using the perfused mouse pancreas. Briefly, the aorta was ligated above the coeliac artery and below the superior mesenteric artery and then cannulated. The pancreas was perfused with KRB containing glucose and CYN154806 at a speed of 0.24 ml/min using an Ismatec Reglo Digital MS2/12 peristaltic pump. The perfusate was maintained at 37C using a Warner Instruments temperature control unit TC-32 4B in conjunction with a tube heater (Warner Instruments P/N 64-0102) and a Harvard Apparatus heated rodent operating table. The effluent was collected in intervals of 1 1 min. Samples were subsequently stored at -80C. Glucagon content in perfusate were measured using U-plex glucagon ELISA (Meso Scale Discovery), according to the manufacturers protocol. Intracellular [Ca2+] measurements [Ca2+]i measurements were performed as described previously35. Islets were imaged in a heated chamber at 37oC placed on an inverted LSM510 confocal microscope (Zeiss; Oberkochen, Germany) using a 40X oil objective (NA1.4). The pinhole diameter was kept constant, and frames of 256×256 pixels were taken every 1-3 s. Parallel measurement of membrane potential and [Ca2+]i The electrophysiological measurements were performed in intact islets essentially using the perforated-patch whole-cell technique in the voltage- or current-clamp modes in -cells. Parallel measurements of [Ca2+]i and membrane potential were performed using an Axioskop 2FS microscope (Zeiss, Oberkochen, Germany) equipped with a 40x/0.8 objective, Lambda DG-4 exciter (Sutter Instruments, USA) and Orca-R2 cooled CCD camera (Hamamatsu, Japan). Images were acquired using an open-source Micromanager software (developed at Ron Vales lab, UCSF, San Francisco, USA) and processed using ImageJ. Data analysis was performed in Igor Pro (Wavemetrics). Intracellular Na+ and pH measurements Time-lapse imaging of [Na+]i in dispersed mouse islets was performed.in of 36 insulin-responsive -cells from 2 mice. islets. Three examples of recordings of [Ca2+]i in individual -cells within intact pancreatic islets are shown in Fig. 1a. The glucose responsiveness was variable: spontaneous [Ca2+]i oscillations were observed in 273% of the cells at 1 mM glucose, which increased to 487% (p 0.05 vs 1 mM) at 4 mM and 825% at 20 mM glucose (p 0.001 vs 1 mM; 79 cells in 7 islets from 7 mice). Increasing glucose from 1 to 4 and 20 mM stimulated Sst release by 100% and 1000%, respectively (Fig. 1b), responses that were associated with comparable increases in the frequency of the [Ca2+]i oscillations (Fig. 1c). When applied at 1 mM glucose, the KATP channel blocker tolbutamide (0.2 mM) produced a 5-fold increase in the frequency of the [Ca2+]i oscillations (Fig. 1d and Extended Data 1a). Conversely, the KATP channel activator diazoxide and the L- and R-type Ca2+ channel blockers isradipine and SNX-482, respectively, abolished or reduced glucose-induced [Ca2+]i oscillations in most -cells and strongly inhibited Sst secretion (Fig. 1e-g and Extended Mycophenolic acid Data 1 and ?and2).2). Sst secretion involves intracellular Ca2+ release by a mechanism sensitive to ryanodine and thapsigargin8 (Extended Data 2a). The inhibitory effect of thapsigargin on Sst secretion correlated with an average 40% decrease in the frequency of the [Ca2+]i oscillations (Extended Data 2e). Open in a separate window Figure 1 Regulation of somatostatin secretion by Ca2+.but in the presence of 100 nM insulin and 1 nM dapagliflozin (Dapa). The dotted line shows data for insulin-unresponsive cells. Data in are mean values S.E.M. in of 36 insulin-responsive -cells from 2 mice. but measuring the effect of lowering [K+]o from 4.7 to 2.7 mM. Bar graph in ((which encodes SGLT2) is low in mouse -cells and that of (encoding SGLT1) is higher (although still lower than transcripts encoding GLUT1-3; see Supplementary Table 1 and 24). The low expression of SGLT1/2 would be in agreement with the small size of the current (~1 pA) in -cells inhibited by high (M) concentrations of dapagliflozin14. In kidney cells, insulin selectively activates SGLT2 (via an effect involving protein phosphorylation) with little effect on SGLT125 but Mycophenolic acid it remains possible that SGLT1 is definitely insulin-sensitive in -cells. Dapagliflozin has been reported to stimulate glucagon secretion both in -cells, the possibility that the dapagliflozin-induced suppression of Sst secretion displays an off-target SGLT2-self-employed effect remains possible, similar to what was recently reported for the related compound canagliflozin28. Ultimately, to conclusively demonstrate that SGLT1 or 2 are practical in -cells, studies would need to become carried out using -cell-specific ablation of and/or measurements of glucagon secretion were performed using the perfused mouse pancreas. Briefly, the aorta was ligated above the coeliac artery and below the superior mesenteric artery and then cannulated. The pancreas was perfused with KRB comprising glucose and CYN154806 at a rate of 0.24 ml/min using an Ismatec Reglo Digital MS2/12 peristaltic pump. The perfusate was managed at 37C using a Warner Devices temperature control unit TC-32 4B in conjunction with a tube heater (Warner Devices P/N 64-0102) and a Harvard Apparatus heated rodent operating table. The effluent was collected in intervals of 1 1 min. Samples were subsequently stored at -80C. Glucagon content material in perfusate were measured using U-plex glucagon ELISA (Meso Level Discovery), according to the manufacturers protocol. Intracellular [Ca2+] measurements [Ca2+]i measurements were performed as explained previously35. Islets were imaged inside a heated chamber at 37oC placed on an inverted LSM510 confocal microscope (Zeiss; Oberkochen, Germany) using a 40X oil objective (NA1.4). The pinhole diameter was kept constant, and frames of 256×256 pixels were taken every 1-3 s. Parallel measurement of membrane potential and [Ca2+]i The electrophysiological measurements were performed in intact islets essentially using the perforated-patch whole-cell technique in the voltage- or current-clamp modes in -cells. Parallel measurements of [Ca2+]i and. em b /em , Average islet glucagon content material from non-diabetic and T2DM organ donors (n=41 non-diabetic and n=12 T2DM donors). and advertising intracellular Ca2+-induced Ca2+ launch (CICR). This mechanism also becomes triggered when [Na+]i is definitely elevated following a inhibition of the plasmalemmal Na+-K+ pump by reductions of the extracellular K+ concentration emulating those produced by exogenous insulin but low manifestation of and was also recognized. We correlated Sst launch and -cell cytoplasmic Ca2+ ([Ca2+]i) in Sst-Cre-GCaMP3 islets. Three examples of recordings of [Ca2+]i in individual -cells within intact pancreatic islets are demonstrated in Fig. 1a. The glucose responsiveness was variable: spontaneous [Ca2+]i oscillations were observed in 273% of the cells at 1 mM glucose, which increased to 487% (p 0.05 vs 1 mM) at 4 mM and 825% at 20 mM glucose (p 0.001 vs 1 mM; 79 cells in 7 islets from 7 mice). Increasing glucose from 1 to 4 and 20 mM stimulated Sst launch by 100% and 1000%, respectively (Fig. 1b), reactions that were associated with similar raises in the rate of recurrence of the [Ca2+]i oscillations (Fig. 1c). When applied at 1 mM glucose, the KATP channel blocker tolbutamide (0.2 mM) produced a 5-fold increase in the frequency of the [Ca2+]i oscillations (Fig. 1d and Extended Data 1a). Conversely, the KATP channel activator diazoxide and the L- and R-type Ca2+ channel blockers isradipine and SNX-482, respectively, abolished or reduced glucose-induced [Ca2+]i oscillations in most -cells and strongly inhibited Sst secretion (Fig. 1e-g and Extended Data 1 and ?and2).2). Sst secretion entails intracellular Ca2+ launch by a mechanism sensitive to ryanodine and thapsigargin8 (Extended Data 2a). The inhibitory effect of thapsigargin on Sst secretion correlated with an average 40% decrease in the rate of recurrence of the [Ca2+]i oscillations (Extended Data 2e). Open in a separate window Number 1 Rules of somatostatin secretion by Ca2+.but in the presence of 100 nM insulin and 1 nM dapagliflozin (Dapa). The dotted collection shows data for insulin-unresponsive cells. Data in are imply ideals S.E.M. in of 36 insulin-responsive -cells from 2 mice. but measuring the effect of decreasing [K+]o from 4.7 to 2.7 mM. Pub graph in ((which encodes SGLT2) is definitely low in mouse -cells and that of (encoding SGLT1) is definitely higher (although still lower than transcripts encoding GLUT1-3; observe Supplementary Table 1 and 24). The low manifestation of SGLT1/2 would be in agreement with the small Mycophenolic acid size of the current (~1 pA) in -cells inhibited by high (M) concentrations of dapagliflozin14. In kidney cells, insulin selectively activates SGLT2 (via an effect involving protein phosphorylation) with little effect on SGLT125 but it remains possible that SGLT1 is definitely insulin-sensitive in -cells. Dapagliflozin has been reported to stimulate glucagon secretion both in -cells, the possibility that the dapagliflozin-induced suppression of Sst secretion displays an off-target SGLT2-self-employed effect remains possible, similar to what was recently reported for the related compound canagliflozin28. Ultimately, to conclusively demonstrate that SGLT1 or 2 are practical in -cells, studies would need to become carried out using -cell-specific ablation of and/or measurements of glucagon secretion were performed using the perfused mouse pancreas. Briefly, the aorta was ligated above the coeliac artery and below the superior mesenteric artery and then cannulated. The pancreas was perfused with KRB comprising glucose and CYN154806 at a rate of 0.24 ml/min using an Ismatec Reglo Digital MS2/12 peristaltic pump. The perfusate was managed at 37C using a Warner Devices temperature control unit TC-32 4B in conjunction with a tube heater (Warner Devices P/N 64-0102) and a Harvard Apparatus heated rodent operating table. The effluent was collected in intervals of 1 1 min. Samples were subsequently stored at -80C. Glucagon content material in perfusate were measured using U-plex glucagon ELISA (Meso Level Discovery), according to the manufacturers protocol. Intracellular [Ca2+] measurements [Ca2+]i measurements were performed as explained previously35. Islets were imaged inside a heated chamber at 37oC placed on an inverted LSM510 confocal microscope (Zeiss; Oberkochen, Germany) using a 40X oil objective (NA1.4). The pinhole diameter was kept constant, and frames of 256×256 pixels were taken every 1-3 s. Parallel measurement of membrane potential and [Ca2+]i The electrophysiological measurements were performed in intact islets essentially using the perforated-patch whole-cell technique in the voltage- or current-clamp modes in -cells. Parallel measurements of [Ca2+]i and membrane potential were performed using an Axioskop 2FS microscope (Zeiss, Oberkochen, Germany) built with a 40x/0.8 objective, Lambda DG-4 exciter (Sutter Instruments, USA) and Orca-R2 cooled CCD camera (Hamamatsu, Japan). Pictures were obtained using an open-source Micromanager software program (created at Ron Vales laboratory, UCSF, SAN FRANCISCO BAY AREA, USA) and prepared using ImageJ. Data evaluation was performed in Igor Pro (Wavemetrics)..