INS-1 832/13 cells (passages 4–30; a gift from Dr. Christopher Newgard, Duke University Medical Center, United States) were cultured in RPMI-1640 media with L-glutamine (Gibco, Amarillo, Texas, United States) containing 10% fetal bovine serum (FBS), 1 mmol/L sodium pyruvate (Invitrogen, Burlington, ON, Canada), 10 mmol/L HEPES and 50 μmol/L β-mercaptoethanol (Sigma, St. Louis, MO, United States) (Krishnamurthy et al., 2008). At 90% confluency, INS-1 were used for experimental studies. For collagen IV matrix study (COL IV), 12- or 96-well tissue culture plates, or cell culture chamber slides (Fisher Scientific, Ottawa, ON, Canada) were precoated with either collagen IV matrix protein at 5 µg per cm² (working solution as 20 μg/ml in 0.05M HCl; Santa Cruz Biotechnology Inc., Dallas, TX, United States) or 1% bovine serum albumin (BSA; Sigma) as control (CTRL). INS-1 cell were placed on precoated plates and cultured in serum-free RPMI medium plus 1% BSA for 24 h. Three to six cell passages were used for each set of experiments, representing n = 3–6. For functional blockade of β1-integrin, INS-1 cells were pretreated with hamster anti-rat β1-integrin antibody (CD29, 5 μg/ml) (anti-β1), hamster IgM isotype-matched negative control (5 μg/ml) (IgM) (BD Biosciences, Mississauga, ON, Canada) or untreated in serum free media as control group (Ctrl) for 1 h prior to being plated on collagen IV pre-coated plates or chamber slides and cultured for 24 h. Cells were harvested and processed for protein extraction or fixed for immunocytochemistry studies.

Cells were plated on 96-well tissue culture plates pre-coated with collagen IV matrix protein or BSA. To analyze adhesion, cells were cultured in serum-free media for 3 h, wells were rinsed twice using 1x PBS to remove non-adhered cells, and six random fields were imaged per well using a Leica DMIRE2 microscope (Leica Microsystems) at 40x magnification. After 24 h, cells were again imaged to analyze cell spreading. Cells that adhered or spread were counted and normalized to control. Data is expressed as fold change versus control. Each experiment was performed in triplicate with six biological repeats per group.

INS-1 cells (1x10⁵) were seeded and cultured on collagen IV or control for 24 h followed by media collection to determine basal insulin secretion. Wells were gently rinsed twice with no glucose RPMI-1640 (Sigma) plus 0.5% BSA followed by incubation in RPMI-1640 plus 0.5% BSA with 2.2 mmol/L glucose then 22 mmol/L glucose for 1 hour each. Media was collected after each treatment to analyze insulin secretion in response to glucose stimulation. Cells were then harvested to determine insulin content.

For time dependent glucose-stimulated insulin secretion (GSIS), INS-1 cells (1.5x10⁵) were plated on cell culture chamber slides pre-coated with collagen IV or BSA for 24 h. Basal media was collected. INS-1 cells were rinsed twice with no glucose RPMI-1640 plus 0.5% BSA, followed by one of four glucose stimulation conditions: followed by one of four glucose stimulation conditions: (1) 2.2 mmol/L glucose for 30 minutes (L30), (2) 22 mmol/L glucose for 5 minutes (H5), (3) 30 minutes (H30), or (4) 60 minutes (H60). Media were collected at each stimulation condition and cells were immediately fixed in 4% PFA followed by immunofluorescent staining for SNARE protein localization analysis.

Insulin concentrations were determined by a Stellux chemiluminescent high range rodent insulin ELISA kit (Alpco, Salem, NH, United States). Static GSIS stimulation index was calculated for each group to account for differences in adhesion and expressed as the ratio of insulin secretion at 22 mM over 2.2 mM glucose stimulation (Krishnamurthy et al., 2008). Insulin content was normalized to total cell protein concentration. Data are expressed as ng/ml or as ng/mg protein (Krishnamurthy et al., 2008). For time dependent GSIS, data are expressed as rate of insulin secretion at ng/ml per minute. Each experiment was performed in technical triplicates with 3–5 biological repeats per group.

Cultured cells were fixed in 4% PFA, embedded in 2% agarose gel and process into tissue blocks (Krishnamurthy et al., 2008). 4 µm sections were taken and stained with appropriate dilutions of primary antibodies as listed in a Supplementary Table S1 followed by incubation with secondary antibodies conjugated with either fluorescein isothiocyanate (FITC) or tetramethyl rhodamine isothiocyanate (TRITC) (Jackson Immunoresearch, West Grove, PA). Nuclei were counterstained with DAPI (Sigma). Positive staining images were captured using a Nikon Eclipse Ti2 confocal microscope (Nikon, Mississauga, ON) set to 60x magnification with oil. Imaging was captured at 6–7 random areas per section with a minimum of three biological repeats per group. Displayed images are representative of staining found in each experimental group.

To assess alterations in the structural organization of focal adhesion contacts, an Actin Cytoskeleton and Focal Adhesion Staining kit was used containing TRITC-conjugated Phalloidin and a monoclonal antibody for Vinculin (Krishnamurthy et al., 2008).

After time-dependent GSIS, INS-1 cells in chamber slides were fixed immediately, incubated in 0.2% Triton for 30 min, then stained and imaged as described above in order to analyze for changes in protein intensity and localization. Displayed images are representative of staining found in each group.

INS-1 cell protein was extracted by sonicating cells in lysis buffer containing Nonident-P40, phenylmethylsolfonyl fluoride, sodium orthovanadate (Sigma) and complete protease inhibitor cocktail tablet (Roche; Mississauga, ON, Canada). Equal amounts (25 µg) of lysate proteins from each experimental group was separated by either 10 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following separation, proteins were transferred to nitrocellulose membrane (Bio-Rad Laboratories Inc.), and membranes were washed in Tris buffer-saline with 0.1% Tween-20 (TBST; Sigma) followed by blocking in 5% nonfat dry milk. Immunoblotting was performed with appropriate dilutions of primary antibodies as listed in Supplementary Table S1 overnight at 4°C, followed by application of appropriate horse-radish peroxidase (HRP)-conjugated secondary antibodies. Proteins were detected by chemiluminescent detection (ECL, PerkinElmer, Waltham, MA, United States). Protein of interest were visualized using a Versadoc version 4.6.9 imaging system, and densitometric quantification of bands was determined by Image Lab software (Bio-Rad Laboratories Inc.). Reference protein (GAPDH) and total proteins were used to normalize protein bands of interest, and data is expressed as fold-change from control (Krishnamurthy et al., 2008; Riopel et al., 2011).

Data are expressed as means ± SEM. Statistical significance was determined using a paired t-test or repeated measures one-way ANOVA followed by Tukey’s post-hoc test, if significant. Differences were considered statistically significant when p < 0.05.

The effect of collagen IV on INS-1 cell adhesion and spreading was first tested, as these are early visual indicators of ECM-cell interactions. Visually and quantitatively, collagen IV provided robust increases in both adhesion and spreading compared to control (p < 0.05, Figures 1A,B). Culturing INS-1 cells on collagen IV provided major differences in focal adhesions compared to control, determined by phalloidin and vinculin staining (Figure 1C). Control cells were round with weak actin and vinculin staining, while cells in the collagen IV group were stretched, and had intense actin at the periphery with increased clusters of vinculin indicating an increase in focal adhesion (Figure 1C). Although basal insulin secretion was not found to differ between groups (Figure 1D), both GSIS stimulation index and insulin content was found to be significantly increased in the collagen IV group versus control (p < 0.05, p < 0.01, Figures 1E,F).

To assess signaling associated with insulin secretion initiated by collagen IV-INS-1 cell interactions, β1-integrin, FAK, and Bag3 were examined. A notable increase in intensity and membrane-associated puncta of β1-integrin was observed in the collagen IV group compared to control (Figure 2A). While no significant changes in total protein levels were found (Figure 2B). FAK is a known downstream signaling molecule from β1-integrin and was found to have elevated phosphorylation in the COL IV group compared to control, although not statistically significant (Figure 2C). Bag3, a protein phosphorylated by FAK and shown to be involved in regulation of insulin exocytosis (Iorio et al., 2015), had a high density of phosphorylated staining (Figure 2D), a minor increase in puncta of total Bag3 staining (Figure 2E), and a 1.4-fold increase in total Bag3 protein levels (Figure 2F) in COL IV cells when compared to control, but statistical significance was not reached due to a large variation between sets of experimental repeats.

To examine whether collagen IV induced increased in insulin secretion is linked to the alteration of SNARE proteins, Munc18-1, Snap25, and Vamp2 were examined. INS-1 cells cultured on collagen IV displayed increased cytoplasmic puncta of Munc18-1 (Figure 3A) with 1.8-fold increase in protein level compared to control (Figure 3B). Cells in the COL IV group also showed increased intensity of Snap25 in the cytoplasm and membrane, relative high staining signals of Vamp2 in the cytoplasm, and an increase in Snap25 and Vamp2 colocalization, as showed in yellow, compared to control (Figure 3C). Both Snap25 and Vamp2 protein levels were elevated in COL IV cells at 1.9- or 1.5-fold when compared to control cells (Figures 3D,E). However, a large variation between sets of experiment repeats made the western blot analysis data not reach statistical significance.

To examine time and glucose-dependent changes of Snap25 and Vamp2 colocalization, double immunofluorescence staining for Snap25 and Vamp2 were performed in INS-1 cells cultured on chamber slides following varied conditions of glucose stimulation. At basal and GSIS conditions, cells cultured on collagen IV showed Snap25 to be more defined membrane staining than control (Figure 4A). A similar colocalization of Snap25 and Vamp2 at basal and following 30 min of low glucose stimulation (L30) was observed in both COL IV and control groups, which was correlated with similar rates of insulin release (Figure 4B). An increase of Snap25 and Vamp2 colocalization at cellular membrane and cytoplasm was found in COL IV cells under 5 min of high glucose stimulation (H5; Figure 4A), with elevated rate of insulin secretion (COL IV 8 ng/mL/minute vs. CTRL 6 ng/mL/minute) (Figure 4B). There was no difference for Snap25 and Vamp2 expression and colocalization at 30 min of high glucose stimulation (H30; Figure 4A) with similar insulin secretion rates (COL IV 1.4 ng/mL/minute vs. CTRL 1.6 ng/mL/minute) between the experimental groups (Figure 4B). At 60 min of high glucose stimulation (H60), cells cultured on COL IV had a higher rate of insulin secretion (1.2 ng/mL/minute) compared to control (0.7 ng/mL/minute) (Figure 4B), along with slightly enhanced Snap25 and Vamp2 expression and colocalization (Figure 4A).

To determine if the enhancement of SNARE protein and insulin secretion in cells cultured on collagen IV was due to β1-integrin signaling, INS-1 cells were pretreated with a β1-integrin blocking antibody. When compared to untreated and IgM-matched controls, INS-1 cells under functional blockade of β1-integrin treatment showed decreased cell adhesion and spreading on collagen IV-coated plates (Figures 5A,B). Blocking β1-integrin also caused significant reduction of basal insulin secretion (∼45%) (p < 0.05, Figure 5C) and GSIS stimulation index (∼50%) (p < 0.01 vs. Ctrl, p < 0.05 vs. IgM, Figure 5D), but no change in cellular insulin content compared to controls (Figure 5E).

Examining INS-1 cell focal adhesions, control cells were highly spread and displayed intense actin filament stress fibers with clusters of vinculin at the periphery (Figure 6A). Anti-β1 cells were rounded in shape with weaker actin staining and membrane-associated vinculin (Figure 6A). Loss of β1 integrin membrane distribution was determined in the anti-β1 group (Figure 6B), along with a reduction of Bag3 phosphorylation signals compared to controls (Figure 6C). Although there was no change of Munc18-1 protein level in anti-β1 cells (Figures 7A,B), anti-β1 treated cells showed reduced Snap25 and Vamp2 staining intensity and protein levels compared to controls (Figures 7C–F). This evidence indicates blocking β1-integrin suppresses collagen IV-INS-1 cell interactions and impairs subsequent downstream signaling and SNARE proteins that are required for insulin exocytosis. Furthermore, anti-β1 cells showed little to no colocalization of Snap25 and Vamp2 during glucose stimulation (Figure 8A), and diminished collagen IV induced high rate of insulin secretion at five or 60 min of high glucose stimulation (H5, H60) when compared to control groups (Figure 8B ).