Orexin A Affects INS-1 Rat Insulinoma Cell Proliferation via Orexin Receptor 1 and the AKT Signaling Pathway

Abstract

Our aim is to investigate the role of the AKT/PKB (protein kinase B) signaling pathway acting via orexin receptor 1 (OX1R) and the effects of orexin A (OXA) on cell proliferation in the insulin-secreting beta-cell line (INS-1 cells). Rat INS-1 cells were exposed to different concentrations of OXA in vitro and treated with OX1R antagonist (SB334867), PI3K antagonist (wortmannin), AKT antagonist (PF-04691502), or negative control. INS-1 amount of cell proliferation, viability and apoptosis, insulin secretion, OX1R protein expression, caspase-3 activity, and AKT protein levels were determined. We report that OXA (10(-10) to 10(-6) M) stimulates INS-1 cell proliferation and viability, reduces the proapoptotic activity of caspase-3 to protect against apoptotic cell death, and increases insulin secretion. Additionally, AKT phosphorylation was stimulated by OXA (10(-10) to 10(-6) M). However, the OX1R antagonist SB334867 (10(-6) M), the PI3K antagonist wortmannin (10(-8) M), the AKT antagonist PF-04691502 (10(-6) M), or the combination of both abolished the effects of OXA to a certain extent. These results suggest that the upregulation of OXA-OX1R mediated by AKT activation may inhibit cell apoptosis and promote cell proliferation in INS-1 cells. This finding provides functional evidence of the biological actions of OXA in rat insulinoma cells.

Figure 1

Effects of OXA on OX1R mRNA and protein expression in INS-1 cells. Cells were exposed to OXA at concentrations of 0 M, 10⁻⁸ M, 10⁻¹⁰ M, and 10⁻⁶ M for 24 h. Another treatment group consisted of 10⁻⁶ M OXA in the presence of the OX1R antagonist SB334867 (OX1Ri) (10⁻⁶ M). The expressions of OX1R mRNA (a) and protein (b) were measured via real-time PCR and western blot analysis. Data are presented as mean ± SEM based on triplicate determinations from a representative experiment. Asterisks indicate significant differences compared to control (*P < 0.05).

Figure 2

Proliferation and viability of INS-1 cells treated with OXA. Cells were treated with OXA at concentrations of 0 M, 10⁻⁸ M, 10⁻¹⁰ M, and 10⁻⁶ M for 24 h. In addition, a separate group of cells was treated with 10⁻⁶ M OXA in the presence of the OX1R antagonist SB334867 (OX1Ri) (10⁻⁶ M) for 24 h. Proliferation and viability were determined by BrdU assays and the MTT test. Data are presented as mean ± SEM based on triplicate determinations from a representative experiment. Asterisks indicate significant differences compared to control (*P < 0.05).

Figure 3

OXA protects INS-1 cells from apoptosis. Cells were exposed to OXA at concentrations of 0 M, 10⁻¹⁰ M, 10⁻⁸ M, and 10⁻⁶ M for 24 h, or cells treated with 10⁻⁶ M OXA in the presence of the OX1R antagonist SB334867 (OX1Ri) (10⁻⁶ M). Apoptosis was assessed with flow cytometry using Annexin V-FITC and PI. Data are presented as mean ± SEM based on triplicate determinations from a representative experiment. Asterisks indicate significant differences compared to control (*P < 0.05).

Figure 4

OXA increases INS-1 cell proliferation via the AKT signaling pathway. INS-1 cells were treated with OXA (10⁻⁶ M) for 20 min in the presence of PF-04691502 (AKTi) (10⁻⁶ M), wortmannin (PI3Ki) (10⁻⁸ M), SB334867 (OX1Ri) (10⁻⁶ M), or the combination of these antagonists. The phosphorylation of AKT (p-AKT) (corresponds to 60 kDa) was normalized against the total protein (t-AKT) activation. The t-AKT protein expression was used as an internal control for equal protein loading. Protein activation was measured by western blot analysis. Data are presented as mean ± SEM based on triplicate determinations from a representative experiment. Asterisks indicate significant differences compared to control samples (*P < 0.05).

Figure 5

Effects of OXA on the proliferation and viability of INS-1 cells via stimulation of the AKT signaling pathway. Cells were exposed to OXA at concentrations of 0 M and 10⁻⁸ M for 24 h in the presence or absence of PF-04691502 (AKTi) (10⁻⁶ M), wortmannin (PI3Ki) (10⁻⁸ M), SB334867 (OX1Ri) (10⁻⁶ M), or a combination of these antagonists. In addition, cells were incubated with AKTi, PI3Ki, OX1Ri without OXA treatment for 24 h. Proliferation and viability were determined by BrdU assay and MTT test. Data are presented as mean ± SEM based on triplicate determinations from a representative experiment. Asterisks indicate significant differences when compared to controls (*P < 0.05).

Figure 6

Effects of OXA on caspase-3 activity in INS-1 cells. Cells were treated with or without OXA (10⁻⁶ M) for 24 h in the presence or absence of PF-04691502 (AKTi) (10⁻⁶ M), wortmannin (PI3Ki) (10⁻⁸ M), SB334867 (OX1Ri) (10⁻⁶ M), or a combination of these antagonists. Caspase-3 activity was assessed using a caspase-3 colorimetric assay kit. Data are presented as mean ± SEM based on triplicate determinations from a representative experiment. Asterisks indicate significant differences as compared to control (**P < 0.01).

Figure 7

Effects of OXA on insulin secretion in INS-1 cells. Cells were exposed to 10⁻⁶ M OXA in the presence or absence of PF-04691502 (AKTi) (10⁻⁶ M), wortmannin (PI3Ki) (10⁻⁸ M), or SB334867 (OX1Ri) (10⁻⁶ M), or a combination of these antagonists. Insulin content was assessed via ELISA. Data are presented as mean ± SEM based on triplicate determinations from a representative experiment. Asterisks indicate significant differences compared to control (**P < 0.01).