Enhanced expression of β cell CaV3.1 channels impairs insulin release and glucose homeostasis

Significance We reveal that increased expression of CaV3.1 channels in rat islets selectively impairs first-phase glucose-stimulated insulin secretion. This deterioration is recapitulated in human islets. Its causal role in diabetes development is clearly manifested in an in vivo diabetic model. Mechanistically, this is due to reduction of phosphorylated FoxO1 in the cytoplasm, elevated FoxO1 nuclear retention, and decreased syntaxin 1A, SNAP-25, and synaptotagmin III in a CaV3.1 channel- and calcineurin-dependent manner. Our findings suggest that elevated expression of CaV3.1 channels in pancreatic islets drives FoxO1-mediated down-regulation of exocytotic proteins resulting in the diabetic phenotypes of impaired insulin secretion and aberrant glucose homeostasis. This causal connection pinpoints β cell CaV3.1 channels as a potential druggable target for antidiabetes therapy.

with a mixture of 40% oxygen and 2.5% isoflurane. Its cornea was punctured with a sharp needle under a stereomicroscope. Subsequently, the blunt eye cannula was gently inserted through the punctured hole into the anterior chamber of the rat eye. Each STZ-treated rat was transplanted with 500 islets, equally divided between the two anterior chambers. Fasting blood glucose levels were monitored before and after STZ injection and after islet transplantation using an Accu-Chek™ blood glucose meter (Roche Diagnostics, Indianapolis, IN).
To perform in vivo confocal imaging, the rat was anesthetized with a mixture of 40% oxygen and 2.5% isoflurane at 4 weeks after islet transplantation and placed on a heating pad to keep its body temperature at 37°C. The head of the anesthetized rat was immobilized in a head holding adaptor (SG-3N; Narishige, Tokyo, Japan) that tilts the eye containing the engrafted islets to a proper orientation. For visualization of vascular structures, the rat was intravenously injected with 70 kDa dextran-conjugated Texas Red (Molecular Probes, Eugene, OR). GFP-positive islet cells and vascular structures filled with dextran-conjugated Texas Red within the engrafted islets were visualized under DMIRBE microscope equipped with a Leica TCS-SP2 confocal laser-scanner. GFP and Texas Red were excited by 488 nm and 595 nm laser lines and the resultant emissions were captured using a long-distance water-dipping lens (Leica HXC APO 10x/0.3W) at 505-530 nm and 610-630 nm, respectively. Reflection images of islets were obtained with 633 nm illumination and collection of light between 630 and 636 nm.
Electrophysiology. COS-7 cells and rat islet cells were subjected to single channel and wholecell patch-clamp measurements following infection with Ad-EGFP and Ad-EGFP-Ca V 3.1. Cellattached and conventional whole-cell patch-clamp configurations were employed. Single channel and whole-cell currents were recorded with an Axopatch 200B amplifier (Molecular Devices, Foster City, California) and an EPC-9 patch clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany), respectively, at room temperature (about 22°C). Recording electrodes were made from borosilicate glass capillaries, fire-polished and coated with Sylgard close to their tips. Some of them were filled with a solution containing (in mM) 110 BaCl 2 , 10 TEA-Cl, and 5 HEPES (pH 7.4 with Ba(OH) 2 for single channel measurements. Others were filled with a solution composed of (in mM) 150 N-methyl-D-glucamine, 125 HCl, 10 EGTA, 1.2 MgCl 2 , 3 MgATP, and 5 HEPES (pH 7.15) for whole-cell current recordings. Electrode resistance ranged between 4 and 6 MΩ when they were filled with electrode solutions and immersed in bath solutions. The electrode offset potential was corrected in bath solutions prior to gigaseal formation. Single-channel recordings were performed with cells bathed in a depolarizing external recording solution, containing (in mM) 125 KCl, 30 KOH, 10 EGTA, 2 CaCl 2 , 1 MgCl 2 , and 5 HEPES-KOH (pH 7.15). This solution was used to bring the intracellular potential to 0 mV. For whole-cell current measurements, the cells were bathed in a solution containing (in mM) 148 tris(hydroxymethyl)aminomethane, 5.6 KCl, 1.2 MgCl 2 , 10 CaCl 2 and 5 HEPES (pH 7.4). Acquisition and analysis of single channel and whole-cell current data were done using the software program pCLAMP 10 (Axon Instruments) and the software program PatchMaster/FitMaster (HEKA), respectively.

SDS-PAGE and Immunoblot
Analysis. INS-1E cells and rat islets were lysed in a lysis buffer (pH 7.5) consisting of (in mM) 50 HEPES, 150 NaCl, 1 EGTA, 1 EDTA, 10% glycerol, 1% triton X-100, 1 PMSF and a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) following different treatments. The lysates were centrifuged at 800 X g for 10 min at 4°C to remove cell debris and nuclei. The protein concentration of the resulting samples was determined with Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). The lysates were denatured by heating at 96°C for 4 min in SDS sample buffer and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. 60 µg of proteins were separated in discontinuous gels consisting of a 4% acrylamide stacking gel (pH 6.8) and an 10% acrylamide separating gel (pH 8.8). The separated proteins were then electroblotted to hydrophobic polyvinylidene difluoride membrane (Hybond-P; GE Healthcare, Uppsala, Sweden). The blots were blocked by incubation for 1 h with 5% non-fat milk powder in a washing buffer, containing (in mM) 20 tris(hydroxymethyl)aminomethane, 150 NaCl and 0.05% Tween 20 (pH 7.5). They were then incubated overnight at 4ºC with rabbit polyclonal antibodies to synaptotagmin III (1:1000; Catalog number: ab81538, Abcam, Cambridge, UK) and phospho- , respectively, at 4ºC overnight or room temperature for 2 h. The specimens were thereafter treated with goat anti-rabbit IgG or antimouse IgG1 coupled to Alexa 633 or 546 (1:1000, Thermo Fisher, Waltham, MA) at room temperature for 1 h. After antibody labeling, specimens were mounted in ProLong ® Gold Antifade mountant with DAPI (Thermo Fisher, Waltham, MA) to prevent photobleaching of fluorophores conjugated to secondary antibodies and to counterstain cell nuclei. Omission of the primary antibodies, incubation with nonimmune IgG from corresponding species or preabsorption of antigen peptides were used as controls. The dilution and incubation period of primary and secondary antibodies and other procedures were kept consistent among batches of experiments. The labeled cells were visualized with a Leica TCS SP8 X confocal laser scanner equipped with a white light and 405 nm pulsed laser and connected to a Leica DMi8 microscope. Alexa 633, Alexa 546 and DAPI were excited by 633 nm, 546 nm and 405 nm laser lines, respectively, and the resultant emissions were captured using a Leica PL APO CS2 63x/1.30 GLYC objective at 645-730 nm, 560-630 nm and 410-440 nm, respectively. The same parameters and settings were used for imaging the labeled cells from batch to batch. Confocal images and their voxel intensities were analyzed with Volocity software (PerkinElmer). The ratio of the mean voxel intensity of nuclear FoxO1 immunofluorescence image to that of cytoplasmic FoxO1 immunofluorescence image was used as the index of FoxO1 nuclear retention and the mean voxel intensity of syntaxin 1A, SNAP-25, synaptotagmin III and VII immunofluorescence images was used as the readout of expression levels of these proteins.
Islet Perifusion and Insulin Secretion Assay. Groups of 50 isolated rat islets untreated or infected with Ad-EGFP or Ad-EGFP-Ca V 3.1 were transferred to 0.27-ml columns containing Bio-Gel P4 polyacrylamide beads (Bio-Rad, Hercules, CA) and perifused at a flow rate of 150 µl/min at 37°C. Effluent perifusate samples were collected every min for the duration of the experiments. Perifusion solution consisted of (in mM) 119 NaCl, 4.6 KCl, 2 CaCl 2 , 1 MgSO 4 , 0.15 Na 2 HPO 4 , 0.4 KH 2 PO 4 , 5 NaHCO 3 and 20 HEPES (pH 7.4) as well as 0.1% bovine serum albumin supplemented with 3 mM or 16.7 mM glucose. Insulin concentrations in the collected samples were determined by using separation-free ArcDia TM TPX assay technique and normalized to islet DNA contents by using DNA quantification kit (Sigma-Aldrich).
Data Analysis. All data are presented as mean ± SEM. One-way ANOVA followed by least significant difference (LSD) test and Student's t test were used to detect statistically significant differences between multiple treatments and between two treatments, respectively. The significance level was set to < 0.05.