Deletion of Ia-2 and/or Ia-2β in mice decreases insulin secretion by reducing the number of dense core vesicles.

Insulin secretion, content and DCV number were evaluated in islets from single knockout (IA-2 KO, IA-2β KO) and double knockout (DKO) mice by a variety of techniques including electron and two-photon microscopy, membrane capacitance, Ca 2+ currents, DCV half-life, lysosome number and size and autophagy.

Methods Mice

KO mice were prepared as described previously [ 11 – 13 ]. Because female IA-2 −/− /IA-2β −/− (DKO) mice are infertile [ 14 ], male DKO mice were bred to female IA-2 −/− /IA-2β +/− mice to generate DKO mice. Animals used in this study were produced in our institute animal core facility. All protocols were approved by NIDCR animal care and use committee.

Islet isolation

Islets from age (3–4 months old) and gender-matched mice were isolated as described previously [ 17 , 18 ]. Briefly, mice were anesthetized by intraperitoneal injection with ketamine (50 mg/kg). Collagenase solution (Sigma, Saint Louis, MO) was injected into the bile duct to inflate the pancreas. After digestion, islets were manually selected and washed in Krebs-Ringer HEPES buffer, and cultured overnight in RPMI-1640 medium (Invitrogen, Carlsbad, CA) before further experiments.

Electron microscopy

Isolated islets were fixed, sectioned [ 18 , 19 ] and analyzed at a primary beam voltage of 120 kV using a CM120 transmission electron microscope (FEI, Hillsboro, OR) and a Gatan cooled 1k × 1k CCD camera (Gatan, Pleasanton, CA). Images were recorded with Digital Micrograph (Gatan) and montaged to evaluate the overall organization of single cells. Each image was quantified by three individuals. Total DCV and lysosomal numbers and the number of DCVs below the plasma membrane were determined using NIH ImageJ.

Two-photon excitation imaging of exocytosis

Exocytosis of DCVs was visualized in living islets with a solution containing the fluid-phase tracer sulforhodamine B (0.7 mmol/l) and two-photon excitation imaging [ 20 , 21 ]. Exocytosis in response to 20 mmol/l glucose was measured within an arbitrary area (800 μm 2 ) of the islets. Imaging was acquired at 1 Hz using an inverted, laser-scanning microscope (FV1000 and I × 81, Olympus, Tokyo) equipped with a water-immersion objective (UPlanApo60xW/IR; N.A. 1.2, Olympus) and a femtosecond laser (MaiTai, Spectra Physics, Mountain View, CA). Sites of exocytosis were detected manually. Electrophysiological studies Details of measurements of intracellular free Ca 2+ concentration [ 22 , 23 ], voltage-gated Ca 2+ current [ 22 , 23 ], and membrane capacitance of β cells [ 24 ] are provided in the Supplemental Methods . Insulin content and secretion Groups of 25 islets on a 62-μm monofilament nylon mesh inside a 13-mm Swinnex chambers (Millipore, Bedford, MA) were perifused at 0.5 ml/min with KRB buffer containing 2.8 or 16.7 mmol/l glucose. Aliquots of perfusate (0.5-ml) were collected at different times over one hour and stored at −80 °C for measurements (Insulin RIA kit, Linco, St. Charles, Missouri). Insulin content in acidic alcohol-extracted islets was determined by RIA. Proinsulin content was determined by ELISA with anti-mouse proinsulin antibody CCI-17 (HyTest, Turku, Finland). Total protein level was measured by the MicroBCA Protein Assay (Pierce, Rockford, IL). Cathepsin D Two hundred freshly isolated islets were washed in Hank’s solution and dissolved by sonication in 200 μl acetate-EDTA buffer (1.1 mmol/l EDTA, 5 mmol/l acetate, pH 5.0). Aliquots were used to determine lysosomal enzyme activity using a Cathepsin D Assay Kit (Sigma). Autophagy Islets were cultured overnight before infection with adenovirus-GFP or LC-3::GFP (gift from Dr. Cindy Miranti). Control cells were infected with GFP alone (Ad-GFP). Islet cells were infected for 24 h before washing and cultured for an additional 24 h. Immunofluorescence with LC-3 antibody (Clone 4E12, MBL, Woburn, MA) was detected by confocal microscopy (Leica TCS SP2 microscope, Wetzlar, Germany). To quantitate the extent of autophagy, number of LC3::GFP punctate in each cell was counted using MetaMorph software (Molecular Devices).