Effect of Glucose and Insulin on Beta-defensins in Keratinocytes

Objective: Human beta-defensin(hBD)s belong to a group of antimicrobial peptides expressed in epithelial cells. Diabetes is a risk factor in oral infections including periodontitis. The hyperglycemia and insulin deficiency/insensitivity may affect hBDs expression. Therefore, the present study investigated the effect of glucose and insulin on the expression of hBD-1,-2 and -3. Methods: HSY cells, from a human salivary duct cell line, were grown in DMEM supplemented with 10% fetal bovine serum. To examine the effect of glucose and/or insulin on the expression of hBDs mRNAs and proteins, the cells were incubated with 5.5, 10, 15 and 50mM of glucose and/or with 100, 150 and 200nM of insulin for 6hr. The cells incubated without glucose or insulin were used as controls. Expression of hBD1~3 in HSY cells were observed by RT-PCR and quantitative RT-PCR using TaqMan probes. The data was analyzed using one-way ANOVA. To evaluate peptide expression level of hBDs, an ELISA assay was carried out. Cell free supernatant and cell lysate from each of cultures and un-stimulated control culture were analyzed for the level of hBD-1, hBD-2 and hBD-3 peptides, using an ELISA. We combined a sandwich ELISA, the best commercially available capture and detection antibodies (Peprotech, NJ). Fold induction represent means ± SD of triplicate experiments. P values were calculated by student's t test. Results: No significant differences are observed in the expression of hBDs between any concentrations of glucose. Expression levels of hBDs in 200nM of insulin were significantly higher than those in the controls. Combination of 5.5mM of glucose and 200nM of insulin stimulated the highest levels of the hBDs in each experiment. Conclusion: The results indicated that optimal concentration of glucose and insulin might be needed to maintain a high expression level of hBDs.