Mouse model of KATP-induced neonatal diabetes mellitus
All experiments were performed in compliance with the relevant laws and institutional guidelines, and were approved by the Washington University Animal Studies Committee. The generation of mice expressing Rosa26-Kir6.2
[K185Q,ΔN30] mutant-transgene with green fluorescent protein (GFP) is described in detail elsewhere [5
]. These mice were crossed with mice expressing Pdx1PB-CreER
] to generate pancreatic beta cell-specific double transgenic (DTG) mice. Littermate wild-type and single transgenic mice which have normal blood glucose levels and insulin secretion were used as controls [5
]. At 8 weeks of age, control and DTG mice received five consecutive daily doses of tamoxifen (50 mg/g body weight, experimental days 0–4). DTG protected mice received a transplant of islets removed from wild-type mice. The transplant was placed under the kidney capsule 2 days prior to the initial tamoxifen injection, following described procedures [5
]. Blood glucose measurements were taken daily using a glucometer (Elite XL; Bayer, Leverkusen, Germany). Mice were killed at experimental days 24 to 28 for islet isolation.
Islet isolation and dispersal
Islets were isolated by collagenase digestion as described in [10
] and maintained in islet medium (RPMI medium containing 10% fetal bovine serum (vol./vol.), 11 mmol/l glucose, 100 U/ml penicillin, 100 μg/ml streptomycin) at 37°C under humidified 5% CO2
for 24 to 48 h before imaging. To obtain dispersed beta cells, islets were washed in Ca2+
-free HBSS, then incubated at 37°C with trypsin/EDTA (both 0.01%, wt/vol.) for 5 min. Trypsin-treated islets were then dispersed by gently re-suspending in islet medium. Dispersed beta cells were plated on glass coverslips in multi-well plates and maintained for 24 to 48 h at 37°C and 5% CO2
Insulin secretion assays
Following overnight incubation in low glucose (5.6 mmol/l) CMRL-1066 medium, islets (ten per well) or dispersed cells (from ten islets per well) were pre-incubated for 30 min at 37°C in glucose-free CMRL-1066 plus 3 mmol/l glucose, then incubated for 60 min at 37°C in CMRL-1066 plus different glucose concentrations, 1 μmol/l glibenclamide and 30 mmol/l KCl as indicated. When 18-α-glycyrrhetinic acid (αGA) treatment was performed, islets were pre-incubated for 30 min with 10 or 50 μmol/l αGA, prior to incubation with 10 or 50 μmol/l αGA plus different glucose concentrations. The medium was then removed and assayed for insulin release. Islets were disrupted using ethanol-HCl extraction and sonicated on ice prior to estimation of insulin content. Insulin secretion and content were measured in triplicates using rat insulin radioimmunoassay according to manufacturer's procedure (Linco, St Charles, MO, USA).
Widefield, confocal and two-photon microscopy
To measure cellular [Ca2+
response and dynamics, isolated islets or dispersed beta cells were stained at room temperature for 1 to 3 h with 4 μmol/l FuraRed-AM and/or 4 μmol/l Fluo4-AM (Invitrogen, Carlsbad, CA, USA) in imaging medium (125 mmol/l NaCl, 5.7 mmol/l KCl, 2.5 mmol/l CaCl2
, 1.2 mmol/l MgCl2
, 10 mmol/l HEPES, 2 mmol/l glucose, 0.1% BSA (wt/vol.), pH 7.4). Intact islets were imaged in a polydimethylsiloxane microfluidic device [7
], facilitating stable imaging and rapid reagent change. Dispersed beta cells were imaged in labtex dishes (Nunc, Roskilde, Denmark). Islets or beta cells were imaged in a humidified chamber maintained at 37°C using a slit-scanning microscope (LSM5Live, Zeiss, Jena, Germany) with a ×20 0.8NA Fluar objective. Images were acquired for 150 or 300 s at 10 min after glucose stimulation, 15 min after application of gap-junction inhibitor or immediately after sulfonylurea or KCl application. Fluo4- or GFP-fluorescence was detected using a 488 nm diode laser for excitation and a 495 to 555 nm band-pass filter for emission. FuraRed fluorescence was detected using the same diode laser and a 650 nm long-pass filter for emission. There was no detectable cross-talk between FuraRed and Fluo4/GFP channels. Background fluorescence was measured from unstained islets.
To measure absolute [Ca2+]i levels, islets were stained for 30 min with 2 μmol/l Fura2-AM and imaged at 37°C in a microfluidic device on a widefield microscope (TE-300; Nikon, Tokyo, Japan) with a 20× 0.45NA Fluar objective. Images were acquired 10 min after glucose stimulation. Islets were sequentially excited at 340 and 380 nm (±10 nm band-pass filter), and fluorescence detected with a 470 to 550 nm band-pass filter. Background fluorescence was measured from unstained islets.
To measure NAD(P)H, islets were imaged at 37°C in a microfluidic device on an LSM710 microscope (Zeiss) with a ×40 1.2NA apochromatic water-immersion objective. NAD(P)H autofluorescence was detected using a 710 nm mode-locked Ti:sapphire laser oscillator (Coherent, Santa-Clara, CA, USA) for two-photon excitation, and custom 380 to 500 nm band-pass filter (Chroma, Rockingham, VT, USA) and non-descanned detector for emission. GFP fluorescence was detected using a 488 nm Ar+ laser line for excitation and a 505 nm long-pass filter for emission. Z-stacks of six images were acquired at 2 μm spacing. No GFP fluorescence was detected in the NAD(P)H channel. All microscope and laser settings were kept constant between measurements.
To assess changes in mitochondrial membrane potential, islets were stained for 30 min with 50 nmol/l tetramethylrhodamine ester (TMRE), then imaged at 37°C in a microfluidic device on a LSM710 microscope with a ×40 1.2NA apochromatic water-immersion objective. Images were acquired 20 min after glucose stimulation. TMRE fluorescence was detected using a 561 nm diode-pumped solid-state laser for excitation and a 580 to 680 nm band-pass filter for emission. Z-stacks of six images were acquired at 2 μm spacing.
Image analyses were performed in Matlab (Mathworks, Natick, MA, USA) and Image Examiner (Zeiss). To calculate fold changes in Fluo4/FuraRed ratio, Fluo4 and FuraRed fluorescence intensities were averaged across each islet and background subtracted. Fluo4/FuraRed intensity ratio was taken and averaged over the measurement time (150 s). For each islet, the mean Fluo4/FuraRed ratio was normalised to the ratio measured at 2 mmol/l glucose. For Fura2 measurements, 340 and 380 nm intensities were background-subtracted and averaged across each islet. Mean [Ca2+]i levels were calibrated from background-subtracted 340:380 nm intensity ratio using a kit (Fura2 Calcium Calibration Kit; Invitrogen)
NAD(P)H and TMRE fluorescence were averaged across each islet and each z-position. To calculate NAD(P)H fluorescence in GFP-positive cells, a GFP threshold intensity was calculated from the mean autofluorescence level in the GFP channel from GFP-negative control islets. This threshold value excludes >99% of GFP-negative cells as measured in control islets. For GFP-positive and GFP-negative cells, NAD(P)H fluorescence was averaged over all pixels showing a GFP intensity greater or less than the threshold GFP value respectively.
To estimate the proportion of the islet showing dynamic changes in [Ca2+]i, Fluo4 or FuraRed images were smoothed with a 3×3 filter and the standard deviation of pixel intensity calculated over the acquisition time course. An active area was defined as having a pixel intensity variance >2 SD above silent pixels calculated at 2 mmol/l glucose. To calculate [Ca2+]i dynamics in GFP-positive cells, [Ca2+]i was determined in cells showing a GFP fluorescence greater than that measured in control islets as above.
Synchronisation of [Ca2+
dynamics was determined as described [12
] and assessed in cells that showed significant dynamic changes in [Ca2+
(see above). Briefly, the Fluo4 time course in each pixel was cross-correlated with a reference Fluo4 time course averaged over the islet. The maximum cross-correlation value was then taken for each pixel. Areas of the islet showing a maximum cross-correlation >0.5 represented areas that are synchronised. This synchronised area was expressed as a percentage of the total islet area that showed significant dynamic changes in [Ca2+
Data are presented as mean ± SEM. Unpaired or paired Student's t test was used to assess significance between control and DTG mouse groups. When more than two groups were tested, we assessed significance within each condition tested using ANOVA and Duncan's post hoc test.