We generated heterozygous pancreatic beta cell-specific Rac1-knockout (betaRac1
−/−) mice by crossing Rac1
flox/flox mice, which harbour a modified endogenous Rac1 gene in which exon1 is flanked by loxP sites , with those that express the Cre recombinase gene under the control of the rat insulin-2 gene , as described previously. The mice were backcrossed to mouse strain C57BL/6J for at least eight generations. C57BL/6J mice were obtained from Clea Japan (Tokyo, Japan). Animals were maintained on a 12 h light, 12 h dark cycle and fed normal chow or a high-fat diet containing 30% (wt/wt) fat (14% bovine fat, 14% porcine fat and 2% soybean oil)  from the time of weaning (3 weeks old). Blood glucose and plasma insulin concentrations were determined as described previously [12, 13]. All experiments were performed with male mice. This study was performed in accordance with the guidelines of the Animal Ethics Committee of Kobe University Graduate School of Medicine.
Mice were deprived of food for 16 h. Blood was collected immediately before and 15, 30, 60 and 120 min after the oral administration of glucose (1.5 mg/g) .
Cell culture and transfection of small interfering RNA
At 24 h before transfection, INS-1 cells were re-plated in 12-well plates (60 mm dishes) and transfected with small interfering (si)RNA for Rac1 (SMARTpool; Dharmacon, Lafayette, CO, USA) or scramble controls (Non-Targeting siRNA#2; Dharmacon) with DharmaFECT2 transfection reagent (Dharmacon). After further incubation for 48 h for mRNA, or for 72 h for protein, the cells were harvested for evaluation of insulin secretion and mRNA expression or protein levels.
Assay of insulin secretion from isolated islets
Islets were isolated from 8-week-old mice as described [13, 15]. To assay insulin release, five islets were manually selected, incubated in Krebs–Ringer solution, and stimulated at 37°C for 30 min with various concentrations of glucose, KCl or tolbutamide. The islets were then collected by centrifugation and the supernatant fraction was assayed for insulin secretion using an ELISA kit with a mouse standard (Shibayagi, Gunma, Japan). To measure islet insulin content, islets were solubilised in acid-ethanol solution (74% ethanol, 1.4% HCl [vol./vol.]) overnight at 4°C before insulin ELISA. Insulin secretion from INS-1 cells was measured after a 30 min incubation in Krebs–Ringer–bicarbonate–HEPES buffer (KRBH; 140 mmol/l NaCl, 3.6 mmol/l KCl, 0.5 mmol/l NaH2PO4, 0.5 mmol/l MgSO4, 1.5 mmol/l CaCl2, 2 mmol/l NaHCO3, 10 mmol/l HEPES and 0.1% BSA; pH 7.4) containing the indicated stimulators. The insulin content was determined after extraction with acid ethanol.
Real-time RT-PCR analysis
Total cellular RNA was extracted from INS-1 cells with an RNeasy kit (QIAGEN, Valencia, CA, USA). Real-time RT-PCR analysis of the total RNA extracted from controls and RAC1-knockdown INS-1 cells was performed as described . For real-time quantitative reverse transcription and PCR analysis, cDNA synthesised from total RNA was evaluated in a sequence detector (model 7900; Applied Biosystems, Foster City, CA, USA) with specific primers (electronic supplementary material [ESM] Table 1) and SYBR Green PCR Master Mix (Applied Biosystems). The relative abundance of mRNAs was calculated with cyclophilin mRNA as the invariant control.
We prepared lysates of isolated islets or INS-1 cells as described previously [12, 13]. The lysates were probed with antibodies to RAC1 (BD Biosciences Pharmingen, San Diego, CA, USA), pan-actin (Cell Signaling, Danvers, MA, USA), and β-actin (Sigma-Aldrich, St Louis, MO, USA).
Immunostaining and morphometric analysis
The pancreas was immersed in Bouin’s solution, embedded in paraffin and sectioned at 4–5 μm thickness. Sections were stained with antibodies to insulin, glucagon (Dako, Glostrup, Denmark), and RAC1 (BD Biosciences). Immune complexes were detected with secondary antibodies conjugated with either cyanine 3 or FITC (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Quantification of beta cell mass was conducted as described [12, 13].
F-actin staining analysis
F-actin was stained using an F-actin Visualization Biochem Kit (Cytoskeleton, Denver, CO, USA). INS-1 cells cultured on a 12 × 12 mm cover slip were fixed by fixative solution and incubated for 10 min. After washing the cover slip, INS-1 cells were permeabilised by permeabilisation buffer (pH 7.0) and incubated for 5 min. After washing, the F-actin of INS-1 cells was stained with rhodamine phalloidin and incubated for 30 min. For RAC1 staining, an anti-RAC1 antibody (BD Biosciences) was used.
Assay of F-actin/G-actin in vitro
We assayed F-actin/G-actin in INS-1 cells with the G-actin/ F-actin In vivo Assay Kit (Cytoskeleton, Denver, CO, USA). We scraped INS-1 cells in LAS2 buffer (pH 6.9) and lysed them to disrupt the cell membrane. After centrifuging the lysate to a pellet of unbroken cells, we centrifuged the supernatant fraction at 100,000 g to separate F-actin from soluble G-actin. We analysed the supernatant fraction for actin content by immunoblotting with anti-actin antibody.
Perfusion studies of mouse pancreases were performed as described previously . Briefly, overnight-fasted (16 h) male mice (10–12 weeks old) were used. The perfusion protocol began with a 10 min equilibration period using KRBH buffer. The flow rate was 1 ml/min.
Total internal reflection fluorescence microscopy analysis
Mouse pancreatic islets were isolated by the collagenase digestion method and dispersed in Ca2+- and Mg2+-free PBS containing 0.016% trypsin and 0.0066% EDTA at 37°C for 5 min. After washing with ice-cold KRBH, the cells were transferred onto a glass cover slip with high refractive index (refractive index, n 1.8 at 488 nm) (Olympus, Tokyo, Japan) coated with 10 μg/cm2 laminin (Sigma) at 37°C for 3 h. The cells were then infected with adenovirus carrying insulin-Venus at a multiplicity of infection of ten and cultured in DMEM or RPMI-1640 medium supplemented with 10% fetal bovine serum under a humidified condition of 95% air and 5% CO2. After culturing infected primary beta cells for 48 h, the cells on the glass cover slips were pre-incubated on the thermostat-controlled stage at 37°C in KRBH containing 2.8 mmol/l glucose. Then, 30 min after pre-incubation, the cells were incubated with 16.8 mmol/l glucose for 15 min, fixed, immunostained with anti-insulin antibody, and observed by total internal reflection fluorescence microscopy (TIRFM) as described [18, 19].
Data are presented as means ± SEM values and compared by ANOVA. A p value of <0.05 was considered significant.