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

Figures & data

Figure 1. (A) IPGTTs of transgenic (TG, dotted trace; n = 7) and control mice (Ctrl, continuous trace; n = 8) show no significant difference. (B) Insulin secretion from batches of TG or control islets in response to 1 h incubations at 2.8 mmol/L (empty bars; n = 3 experiments) or 16.7 mmol/L glucose (gray bars; n = 3). (C) ChR2-YFP fluorescence in TG islets. Right panel shows an islet at higher magnification and a non-TG (Ctrl) islet stained with DAPI (inset). (D) Representative examples of ChR2-YFP fluorescence in dispersed islets cells stained for insulin (upper panel) or glucagon (lower panel). Merged images are shown to the right. Scale bar is 100 μm. Data are means ± SEM *P < 0.05. (E) Contour plot of fluorescent cell counting using FACS (representative example of β-cell fraction from one animal) with the PE-A signal representing insulin-positive (Ins+) and the FITC-A signal denoting YFP-positive cells (YFP⁺), all in the β-cell gate.

Table 1. FACS results of dispersed islet cells of 4 transgenic animals.

Fraction/Cells in %	Of all islet cells	Beta-cell fraction
YFP^+Ins^+	56.2 ± 6.5	95.6 ± 1.5
YFP^+Ins^-	0.5 ± 0.1	0.2 ± 0.1
YFP^-Ins^+	2.7 ± 0.7	2.1 ± 0.5
YFP^-Ins^-	40.7 ± 5.9	2.1 ± 1.0

Figure 2. (A) Effect of blue LED light illumination on [Ca²⁺]_i in ChR2-expressing transgenic TG islets (n = 11) or ChR-2 negative control islets (control; n = 7) (representative traces shown). Here, each light pulse is 100 ms long (depicted as blue dots, not to scale) and glucose concentration was 2.8 mmol/l. Ten images were acquired in between light pulses. (B) As in (A) but traces show recordings after 15 min pre-incubation with 5 µmol/l isradipine and 100 nmol/l SNX-482 (gray trace; n = 10) compared with untreated TG islets (black trace; n = 11, same trace as in [A]). (C) Changes in [Ca²⁺]_i in response to different light protocols (for details see also Fig. S1). Each blue striped box corresponds to a 1 s stimulation segment during which ten 1 ms (light-colored traces) or ten 10 ms long (dark-colored traces) light pulses were fired at different frequencies: 10 Hz (blue traces; n = 3/8 experiments using 1 ms/10 ms pulses, respectively), 20 Hz (red traces; n = 4/4) or 40 Hz (green traces; n = 4/3). Ten images were acquired in between the 1 s long stimulation segments. (D) As in (c) but using continuous light for 1 s (n = 4) compared with pulsatile stimulation at 40 Hz and 10 ms pulse duration (n = 3). (E) Effect of longer pulsatile illumination on [Ca²⁺]_i. Each thin blue stripe of the stimulation segment corresponds to a 500 ms light pulse with stimulation segment duration as despicted (I: 60, II: 30, and III: 10 s respectively). Images were acquired in between the single light pulses and between the stimulation segments. Representative examples of illumination-evoked currents with 500 ms (F) and 100 ms blue light pulses in single transgenic β-cells. (H) As in (G) but in ChR2-negative control islet cells.

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.

Figure 4. (A) Intraperitoneal glucose tolerance tests in TG (dotted lines) and control animals (solid lines) given a high fat diet for 8 wk (HFD, gray traces; n = 6 TG and 8 control mice) or standard chow diet (SCD, black traces; n = 7 TG and 8 control mice). Differences were assessed by analyzing the AUC. (B) Insulin content in islets from TG or control mice on HFD (gray bars) or SCD (white bars; n = 5 in each group). (C) Representative recordings of Fura-2 fluorescence in response to light stimulation in islets from HFD (gray trace) and SCD mice (black line) at 2.8 mmol/L glucose (n = 4). (D) Insulin secretion from HFD islets with or without 1 h constant light stimulation at 1 mmol/l (empty bars), 5.6 mmol/l (gray) and 16.7 mmol/l glucose (black; n = 5). (E) Insulin release in islets from mice on high-fat diet (n = 3) stimulated with blue LED light (gray bars, +LED) and without LED (white bars; -LED) as well as in the presence (+) or absence (-) of SNX482 (100 nmol/l), isradipine (5 µmol/l). (F) YFP fluorescence intensities in islets from SCD and HFD mice (n = 4). Data are means ± SEM *P < 0.05.