In vitro analysis of the insulin signaling pathway

For in vitro analysis, gingival fibroblasts were isolated from the maxillae gingiva of control rats, as described previously [12]. In brief, the gingiva, including the epithelium and connective tissue, was harvested from the control rats. After dispase I treatment, the connective tissue was minced into small pieces (2 mm²) and incubated in Dulbecco’s modified Eagle’s medium (D-MEM) with 10% fetal bovine serum (FBS) and 3% antibiotics at 37°C. Migrated fibroblast cells were used at passage 3–6 for experiments. The cells were cultured in a medium containing 15 mM glucose as the control or 50 mM glucose as the HG condition on the basis of our previous studies using gingival fibroblast in high-glucose medium [12, 26]. For osmotic control, mannitol was added as control for hyperosmolarity in the high-glucose groups.

To analyze the insulin signaling-related cell migration and proliferation via the PI3K and mitogen-activated protein kinase pathways, phosphorylated Akt (p-Akt) and Erk1/2 (p-Erk1/2) levels were evaluated following insulin stimulation (100 nM) via western blotting. Primary fibroblasts were cultured with the respective glucose concentrations in D-MEM containing 0.5% FBS at 37°C and then treated with insulin (100 nM) for an additional 10 min. Following insulin stimulation, the medium was aspirated and cells were washed with ice-cold PBS. Lysis buffer containing RIPA Buffer (Wako, Tokyo, Japan), protease inhibitor cocktail, and phosphatase inhibitor (Sigma) was added to the culture dish, and the cells were harvested via scraping with a cold scraper. The pellets were sonicated and centrifuged at 13,400 rpm for 10 min at 4°C. The supernatants, 4X Laemmli sample buffer (Bio-rad, Hercules, CA, USA), and 2-mercaptoethanol (Sigma) were mixed and boiled for 5 min at 95°C. Equal amounts of proteins were electrophoresed on Mini-Protean TGX gels (Bio-rad), at a constant voltage of 100 V, and electro-transferred to nitrocellulose membranes (Amersham) at a constant current of 200 mA. Membranes were incubated for blocking at room temperature (25°C) with Blocking Buffer (StartingBlock T20, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min. Thereafter, the membranes were incubated overnight with a 1:1000 dilution of total- (Cell Signaling Technology, Danvers, MA, USA, #9272) and phospho-Akt (Cell Signaling Technology #4060) antibodies, and total- (Cell Signaling Technology #9102) and phospho-Erk1/2 antibodies (Cell Signaling Technology #4370) in Blocking Buffer at 4°C. The membranes were washed with PBST (80 mM Na₂HPO₄, 50 mM NaH₂PO₄, 100 mM NaCl, and 1% Tween 20) for 5 min and incubated with a 1 : 5,000 dilution of donkey antirabbit IgG-HRP (Santa Cruz Biotechnology, sc-2313, Dallas, TX, USA) secondary antibody in Blocking Buffer at room temperature for 1 h. Thereafter, membranes were washed intensively with PBST. The enhanced chemiluminescence reaction generated using the ECL Western Blotting Substrate (Pierce) was analyzed using Ez-Capture MG and analyzed using an imaging software (National Institutes of Health, Bethesda, MD, USA).