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Optogenetic control of insulin secretion in intact pancreatic islets with β-cell-specific expression of Channelrhodopsin-2.

Reinbothe TM, Safi F, Axelsson AS, Mollet IG, Rosengren AH - Islets (2014)

Bottom Line: The effect of ChR2 stimulation with blue LED light was assessed using Ca(2+) imaging and static islet incubations.Moreover, light stimulation enhanced insulin secretion in batch-incubated islets at low and intermediate but not at high glucose concentrations.Glucagon release was not affected.

View Article: PubMed Central - PubMed

Affiliation: a Department of Clinical Sciences; Lund University Diabetes Centre; Malmö, Sweden.

ABSTRACT
Insulin is secreted from the pancreatic β-cells in response to elevated glucose. In intact islets the capacity for insulin release is determined by a complex interplay between different cell types. This has made it difficult to specifically assess the role of β-cell defects to the insulin secretory impairment in type 2 diabetes. Here we describe a new approach, based on optogenetics, that enables specific investigation of β-cells in intact islets. We used transgenic mice expressing the light-sensitive cation channel Channelrhodopsin-2 (ChR2) under control of the insulin promoter. Glucose tolerance in vivo was assessed using intraperitoneal glucose tolerance tests, and glucose-induced insulin release was measured from static batch incubations. ChR2 localization was determined by fluorescence confocal microscopy. The effect of ChR2 stimulation with blue LED light was assessed using Ca(2+) imaging and static islet incubations. Light stimulation of islets from transgenic ChR2 mice triggered prompt increases in intracellular Ca(2+). Moreover, light stimulation enhanced insulin secretion in batch-incubated islets at low and intermediate but not at high glucose concentrations. Glucagon release was not affected. Beta-cells from mice rendered diabetic on a high-fat diet exhibited a 3.5-fold increase in light-induced Ca(2+) influx compared with mice on a control diet. Furthermore, light enhanced insulin release also at high glucose in these mice, suggesting that high-fat feeding leads to a compensatory potentiation of the Ca(2+) response in β-cells. The results demonstrate the usefulness and versatility of optogenetics for studying mechanisms of perturbed hormone secretion in diabetes with high time-resolution and cell-specificity.

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Figure 3. (A) Insulin release in response to 1 h constant blue LED light stimulation (LED) in batch-incubated TG islets (n = 4 replicates) or non-ChR2 expressing control islets (n = 4) at 2.8 mmol/l glucose. (B) Insulin secretion from TG islets at 2.8 mmol/l glucose with or without 1 h constant light stimulation and in the presence or absence of 5 µmol/l isradipine and 100 nmol/l SNX-482 (n = 8). (C) Effect of light stimulation on glucagon release in TG (n = 6) and control islets (n = 6) at 2.8 mmol/l glucose. (D) Insulin secretion with or without 1 h light stimulation at 1 mmol/l (empty bars), 5.6 mmol/l (gray bars) and 16.7 mmol/l glucose (black; n = 7 for all). (E) Effect of different glucose concentrations on basal and light-induced [Ca2+]i concentrations subsequently recorded from the same islets with 5 min incubations in the respective concentration (black continuous trace 1 mmol/l; gray trace 2.8 mmol/l; 5.6 mmol/l in green and 16.7 mmol/l in blue; n = 5 experiments). (F) Same as in (E), but data depicted as changes in [Ca2+]i (Δ[Ca2+]i) to facilitate comparison of the peaks. Data in bar charts are means ± SEM *P < 0.05.
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Figure 3: Figure 3. (A) Insulin release in response to 1 h constant blue LED light stimulation (LED) in batch-incubated TG islets (n = 4 replicates) or non-ChR2 expressing control islets (n = 4) at 2.8 mmol/l glucose. (B) Insulin secretion from TG islets at 2.8 mmol/l glucose with or without 1 h constant light stimulation and in the presence or absence of 5 µmol/l isradipine and 100 nmol/l SNX-482 (n = 8). (C) Effect of light stimulation on glucagon release in TG (n = 6) and control islets (n = 6) at 2.8 mmol/l glucose. (D) Insulin secretion with or without 1 h light stimulation at 1 mmol/l (empty bars), 5.6 mmol/l (gray bars) and 16.7 mmol/l glucose (black; n = 7 for all). (E) Effect of different glucose concentrations on basal and light-induced [Ca2+]i concentrations subsequently recorded from the same islets with 5 min incubations in the respective concentration (black continuous trace 1 mmol/l; gray trace 2.8 mmol/l; 5.6 mmol/l in green and 16.7 mmol/l in blue; n = 5 experiments). (F) Same as in (E), but data depicted as changes in [Ca2+]i (Δ[Ca2+]i) to facilitate comparison of the peaks. Data in bar charts are means ± SEM *P < 0.05.

Mentions: We next assessed whether light could trigger insulin release from TG islets. As constant illumination evoked the same increase of [Ca2+]i compared with pulsatile light (Fig. 2D), we decided to examine the feasibility of using 1 h constant light illumination to stimulate insulin secretion, as that would enable a considerably simpler and more broadly applicable experimental setup. Thus, a blue LED light was attached to tubes with TG islets (see Methods). Interestingly, light stimulation increased insulin secretion at 2.8 mmol/l glucose 2-fold (from 2.8 ± 0.4 to 5.3 ± 1.1 ng/ml, P < 0.05; Fig. 3A). This effect was not observed in non-ChR2 expressing control islets. In agreement with the Ca2+ measurements, blue light did not stimulate insulin release in the presence of L- and R-type Ca2+-channel blockers (Fig. 3B). We found a suppressive effect of the VGCC blockers at 2.8 mmol/l glucose in both TG (3.9 ± 0.4 without blockers vs. 2.5 ± 0.1 ng/ml×h with blockers; P < 0.01, Fig. 3B) and control islets (3.5 ± 0.3 without vs. 2.6 ± 0.2 ng/ml×h with blockers; P < 0.05; Fig. S2). The fold-stimulation of insulin secretion by light was reduced in the presence of blockers (1.3 ± 0.1-fold vs. 1.9 ± 0.2-fold; P < 0.05). We observed no effect of light stimulation on glucagon release (Fig. 3C).


Optogenetic control of insulin secretion in intact pancreatic islets with β-cell-specific expression of Channelrhodopsin-2.

Reinbothe TM, Safi F, Axelsson AS, Mollet IG, Rosengren AH - Islets (2014)

Figure 3. (A) Insulin release in response to 1 h constant blue LED light stimulation (LED) in batch-incubated TG islets (n = 4 replicates) or non-ChR2 expressing control islets (n = 4) at 2.8 mmol/l glucose. (B) Insulin secretion from TG islets at 2.8 mmol/l glucose with or without 1 h constant light stimulation and in the presence or absence of 5 µmol/l isradipine and 100 nmol/l SNX-482 (n = 8). (C) Effect of light stimulation on glucagon release in TG (n = 6) and control islets (n = 6) at 2.8 mmol/l glucose. (D) Insulin secretion with or without 1 h light stimulation at 1 mmol/l (empty bars), 5.6 mmol/l (gray bars) and 16.7 mmol/l glucose (black; n = 7 for all). (E) Effect of different glucose concentrations on basal and light-induced [Ca2+]i concentrations subsequently recorded from the same islets with 5 min incubations in the respective concentration (black continuous trace 1 mmol/l; gray trace 2.8 mmol/l; 5.6 mmol/l in green and 16.7 mmol/l in blue; n = 5 experiments). (F) Same as in (E), but data depicted as changes in [Ca2+]i (Δ[Ca2+]i) to facilitate comparison of the peaks. Data in bar charts are means ± SEM *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Figure 3. (A) Insulin release in response to 1 h constant blue LED light stimulation (LED) in batch-incubated TG islets (n = 4 replicates) or non-ChR2 expressing control islets (n = 4) at 2.8 mmol/l glucose. (B) Insulin secretion from TG islets at 2.8 mmol/l glucose with or without 1 h constant light stimulation and in the presence or absence of 5 µmol/l isradipine and 100 nmol/l SNX-482 (n = 8). (C) Effect of light stimulation on glucagon release in TG (n = 6) and control islets (n = 6) at 2.8 mmol/l glucose. (D) Insulin secretion with or without 1 h light stimulation at 1 mmol/l (empty bars), 5.6 mmol/l (gray bars) and 16.7 mmol/l glucose (black; n = 7 for all). (E) Effect of different glucose concentrations on basal and light-induced [Ca2+]i concentrations subsequently recorded from the same islets with 5 min incubations in the respective concentration (black continuous trace 1 mmol/l; gray trace 2.8 mmol/l; 5.6 mmol/l in green and 16.7 mmol/l in blue; n = 5 experiments). (F) Same as in (E), but data depicted as changes in [Ca2+]i (Δ[Ca2+]i) to facilitate comparison of the peaks. Data in bar charts are means ± SEM *P < 0.05.
Mentions: We next assessed whether light could trigger insulin release from TG islets. As constant illumination evoked the same increase of [Ca2+]i compared with pulsatile light (Fig. 2D), we decided to examine the feasibility of using 1 h constant light illumination to stimulate insulin secretion, as that would enable a considerably simpler and more broadly applicable experimental setup. Thus, a blue LED light was attached to tubes with TG islets (see Methods). Interestingly, light stimulation increased insulin secretion at 2.8 mmol/l glucose 2-fold (from 2.8 ± 0.4 to 5.3 ± 1.1 ng/ml, P < 0.05; Fig. 3A). This effect was not observed in non-ChR2 expressing control islets. In agreement with the Ca2+ measurements, blue light did not stimulate insulin release in the presence of L- and R-type Ca2+-channel blockers (Fig. 3B). We found a suppressive effect of the VGCC blockers at 2.8 mmol/l glucose in both TG (3.9 ± 0.4 without blockers vs. 2.5 ± 0.1 ng/ml×h with blockers; P < 0.01, Fig. 3B) and control islets (3.5 ± 0.3 without vs. 2.6 ± 0.2 ng/ml×h with blockers; P < 0.05; Fig. S2). The fold-stimulation of insulin secretion by light was reduced in the presence of blockers (1.3 ± 0.1-fold vs. 1.9 ± 0.2-fold; P < 0.05). We observed no effect of light stimulation on glucagon release (Fig. 3C).

Bottom Line: The effect of ChR2 stimulation with blue LED light was assessed using Ca(2+) imaging and static islet incubations.Moreover, light stimulation enhanced insulin secretion in batch-incubated islets at low and intermediate but not at high glucose concentrations.Glucagon release was not affected.

View Article: PubMed Central - PubMed

Affiliation: a Department of Clinical Sciences; Lund University Diabetes Centre; Malmö, Sweden.

ABSTRACT
Insulin is secreted from the pancreatic β-cells in response to elevated glucose. In intact islets the capacity for insulin release is determined by a complex interplay between different cell types. This has made it difficult to specifically assess the role of β-cell defects to the insulin secretory impairment in type 2 diabetes. Here we describe a new approach, based on optogenetics, that enables specific investigation of β-cells in intact islets. We used transgenic mice expressing the light-sensitive cation channel Channelrhodopsin-2 (ChR2) under control of the insulin promoter. Glucose tolerance in vivo was assessed using intraperitoneal glucose tolerance tests, and glucose-induced insulin release was measured from static batch incubations. ChR2 localization was determined by fluorescence confocal microscopy. The effect of ChR2 stimulation with blue LED light was assessed using Ca(2+) imaging and static islet incubations. Light stimulation of islets from transgenic ChR2 mice triggered prompt increases in intracellular Ca(2+). Moreover, light stimulation enhanced insulin secretion in batch-incubated islets at low and intermediate but not at high glucose concentrations. Glucagon release was not affected. Beta-cells from mice rendered diabetic on a high-fat diet exhibited a 3.5-fold increase in light-induced Ca(2+) influx compared with mice on a control diet. Furthermore, light enhanced insulin release also at high glucose in these mice, suggesting that high-fat feeding leads to a compensatory potentiation of the Ca(2+) response in β-cells. The results demonstrate the usefulness and versatility of optogenetics for studying mechanisms of perturbed hormone secretion in diabetes with high time-resolution and cell-specificity.

Show MeSH
Related in: MedlinePlus