Effects of voltage-gated sodium channel inhibition on islet [Ca²⁺]<i>_i</i> and hormone secretion.

<p>Gray and black traces represent α- and β-cells, respectively. A, representative intracellular calcium responses to tetrodotoxin (TTX) in an intact mouse islet perifused at 1 mM glucose. Increasing concentrations of TTX were perifused at times indicated by the arrows. TTX stimulates calcium activity in α-cells while having no noticeable effects on β-cells. Fluo-4 intensity is expressed in arbitrary units. The figure is representative of 18 α-cells analyzed from 6 islets harvested from 3 mice. B, effects of TTX on glucagon and insulin secretion from intact perifused islets. Isolated islets were exposed to 1 mM glucose for 30 minutes (from −30 to 0 min). Glucagon and insulin responses were measured for 9 minutes at 1 mM glucose (G1), and then TTX was perifused. Experiment was repeated 6 times, 900 islets from 12 mice were used. Error bars represent the standard error of the mean.</p

Effects of pharmacological modulation of K_ATP channels on islet [Ca²⁺]<i>_i</i> and hormone secretion.

<p>Gray and black traces represent α- and β-cells, respectively. A, representative intracellular calcium responses to K_ATP channel activation by 100 µM diazoxide from an islet perifused at 1 mM (G1). Fluo-4 intensity is expressed in arbitrary units. The figure is representative of 25 α-cells analyzed from 7 islets harvested from 3 mice. B, Effect of diazoxide on hormone secretion from intact perifused islets. Isolated islets were exposed to 1 mM glucose for 30 minutes (from -30 to 0 min). Glucagon and insulin responses were measured for 12 minutes at 1 mM glucose, and diazoxide was perifused for 30 minutes at 100 µM. Experiment was repeated 3 times, 450 islets from 6 mice were used. Error bars represent the standard error of the mean. C, D, and E, representative Fluo-4 responses to K_ATP channel inhibition from an islet perifused at 1 mM glucose. The figure shows 3 different α-cells from the same islet exposed to 100 µM tolbutamide, and is representative of 34 α-cells analyzed from 10 islets harvested from 3 mice. F, glucagon and insulin responses were measured for 9 minutes at 1 mM glucose, and then tolbutamide was perifused at 100 µM. Experiment was repeated 6 times, 900 islets from 12 mice were used. G, representative Fluo-4 responses to 100 µM tolbutamide (TTX) in an intact islet perifused at 12 mM. H, glucagon and insulin secretion from islets perifused at 1 mM glucose and 100 µM tolbutamide for 15 minutes, then glucose concentration was increased to 12 mM. Experiment was repeated 3 times, 450 islets from 6 mice were used.</p

Effects of high-voltage-gated calcium channel inactivation on islet [Ca²⁺]<i>_i</i> and hormone secretion.

<p>A, representative intracellular calcium responses to blockade of N- and L-type calcium channels. Gray traces represent α-cell [Ca²⁺]<i>_i</i>, and black traces indicate β-cell [Ca²⁺]<i>_i</i>. Fluo-4 intensity is expressed in arbitrary units. Calcium responses from two α-cells in the same islet are shown. N- and L-type channel inhibitors (1 µM ω-conotoxin and 20 µM nifedipine, respectively) were perifused. The figure is representative of 15 α-cells from 5 islets isolated from 3 mice. B, increasing concentrations of nifedipine were perifused at times indicated by the arrows. Nifedipine (≤ 10 µM) reduces calcium activity in α-cells without affecting β-cell [Ca²⁺]<i>_i</i>. The figure is representative of 10 α-cells from 3 islets harvested from 3 mice. C, effects of nifedipine on hormone secretion from intact perifused islets. Islets were exposed to 1 mM glucose for 30 minutes (from −30 to 0 min). Glucagon and insulin responses (gray and black traces, respectively) were measured for 15 minutes at 1 mM glucose (G1), and 20 µM nifedipine was added to the perifusion medium for 30 minutes. Experiment was repeated 4 times, 600 islets from 8 mice were used. Error bars represent the standard error of the mean.</p

Glucose effects on hormone secretion from perifused islets.

<p>Isolated islets were exposed to 1 mM glucose for 30 minutes (from −30 to 0 min). Then, both glucagon and insulin responses (gray and black traces, respectively) were measured for 15 minutes at 1 mM glucose (G1). The perifusion was changed to 12 mM (G12) for 15 minutes, and then switched back to 1 mM. Experiment was repeated 3 times, 450 islets from 6 mice were used. Error bars represent the standard error of the mean. To compare the volume of islets with different diameters and volumes, individual islets were mathematically converted to standard islet equivalents (IEQs) with a diameter of 150 µm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047084#pone.0047084-Ricordi1" target="_blank">[44]</a>.</p

Effects of L-arginine on islet NAD(P)H, [Ca²⁺]<i>_i</i>, and hormone secretion.

<p>Gray and black columns and traces represent α- and β-cells, respectively. A, arginine-dependent NAD(P)H responses from intact islets. Islets were perifused at 1 mM glucose (G1) and exposed to step-increases in arginine concentration. Data are normalized to minimal and maximal β-cell NAD(P)H obtained with FCCP and cyanide, respectively. The α-cell NAD(P)H changes to arginine (Arg) were statistically significant (<i>p</i><0.01, 32 α-cells measured from 10 islets, 3 mice) and α-cell NAD(P)H intensity was different from β-cell intensity for each condition tested (<i>p</i><0.01). Error bars indicate the standard error of the mean. B, averaged intracellular calcium responses to arginine. Data are expressed in percent change in Fluo-4 intensity compared to baseline at 1 mM glucose. α-cell responses to arginine were significant at all concentrations (<i>p</i><0.01, n = 42) and β-cell responses were significant at 10 and 20 mM arginine (<i>p</i><0.01, n = 11). C, intracellular calcium responses to 10 mM arginine from an islet perifused at 1 mM glucose (G1). 2 α-cells from the same islet are presented. Fluo-4 intensity is expressed in arbitrary units. The figure is representative of 11 α-cells from 4 islets. D, glucagon and insulin responses were measured for 9 minutes at 1 mM glucose, and then arginine was perifused at 10 mM for 18 minutes. Finally, glucose was added to the perifusion medium at 12 mM for 18 minutes. Experiment was repeated 4 times, 600 islets from 8 mice were used. Error bars represent the standard error of the mean.</p

Differential Stimulation of Insulin Secretion by GLP-1 and Kisspeptin-10 - Figure 1

<p><b>A</b>. Percent of insulin content secreted from intact islets after static incubation at 2.8, 10, or 16.7 mM glucose with and without KP (1 µM, dark gray) or GLP-1 (20 nM, light gray). Secretion from untreated control islets is shown in white. Data are the mean ± S.E. <i>n</i> = 4–19. *(<i>p</i><0.05) and **(<i>p</i><0.001) indicate significance compared to untreated control. <b>B</b>, Glucose-dependent percent change in NAD(P)H from untreated intact islets (circles) and islets treated with KP (1 µM, squares) or GLP-1 (20 nM, triangles) compared to values at 2 mM glucose. Data are the mean ± S.E. <i>n</i> = 9–11. *<i>p</i><0.01.</p

Percent of insulin content secreted from intact islets after static incubation at 2.8, 10, and 16.7 mM glucose with and without treatment.

<p>Untreated control samples are shown in white. <b>A</b>, Percent of insulin content secreted at 2.8, 10, and 16.7 mM glucose concentrations in the presence and absence of GLP-1 (20 nM, light gray), the G_βγ inhibitor, gallein (10 µM, checked), or combination treatment with gallein and GLP-1 (striped). <b>B,</b> Percent of insulin content secreted at 2.8, 10, and 16.7 mM glucose concentrations with GLP-1 (20 nM, light gray), the G_βγ-activating peptide mSIRK (30 µM, checked), or combination treatment with mSIRK and GLP-1 (striped). Data are the mean ± S.E. <i>n</i> = 4–19. *(<i>p</i><0.05) and **(<i>p</i><0.001) indicate significance compared to untreated control, GLP-1 only, or gallein alone.</p

Percent of insulin content secreted from intact islets after incubation at 2.8, 10, or 16.7 mM glucose with and without treatment.

<p>Untreated control samples are shown in white. <b>A</b>, Percent of insulin content secreted at 2.8, 10, and 16.7 mM glucose concentrations in the presence and absence of KP (10 µM, dark gray), gallein (10 µM, checked), or gallein+KP (striped). <b>B</b>, Percent of insulin content secreted at 2.8, 10, and 16.7 mM glucose concentrations with and without KP (10 µM, dark gray), mSIRK (30 µM, checked), or mSIRK+KP (striped). Data are the mean ± S.E. <i>n</i> = 4–19. *<i>p</i><0.05 and **<i>p</i><0.001 compared to untreated control, KP alone, or mSIRK only.</p

Changes in Fluo4 signal in dispersed β-cells recorded at 10 mM glucose in the presence and absence of KP (1 µM) or with GLP-1 (20 nM) to measure the frequency and amplitude of [Ca²⁺]_i oscillations.

<p><b>A & C</b>, Representative oscillations in [Ca²⁺]_i recorded from dispersed β-cells before (dotted line) and after (solid line) treatment with KP (<b>A</b>) or GLP-1 (<b>C</b>). <b>B & D</b>, The normalized [Ca²⁺]_i oscillation frequency measured pre- and post-treatment with KP (<b>B</b>, dark gray) or GLP-1 (<b>D</b>, light gray). Data are normalized to the data collected from the dispersed β-cells prior to ligand treatment (white). Data are the mean ± S.E. <i>n</i> = 4. <i>p</i><0.05.</p

Fluo4 signal recorded at 10 mM glucose in the presence and absence of GLP-1 (20 nM) alone or combined with gallein (10 µM) or mSIRK (30 µM) to detect changes in the frequency and amplitude of [Ca²⁺]_i oscillations.

<p><b>A, C &, E</b>, Representative [Ca²⁺]_i oscillations from intact islets recorded pre- (dotted line) and post-treatment (solid line) with GLP-1 (<b>A</b>), gallein and GLP-1 (<b>C</b>) or mSIRK and GLP-1 (<b>E</b>). <b>B, D, & F</b>, The normalized [Ca²⁺]_i oscillation frequency measured pre- and post-treatment with GLP-1 (<b>B</b>, light gray), gallein and GLP-1 (<b>D</b>, light gray stripes) or mSIRK and GLP-1 (<b>F</b>, light gray checks). Data are normalized to the data collected from the islet prior to ligand/G_βγ modulator treatment (white). Data are the mean ± S.E. <i>n</i> = 4–5. <i>p</i>>0.1.</p