Data are presented as mean SEM based on triplicate determinations from a representative experiment. 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. 1. Introduction Orexin A and orexin B (OXA and OXB), also known as hypocretin-1 Neridronate and hypocretin-2, are peptides that were initially discovered by orphan receptor technologies  and/or substrative cDNA cloning . The two orexins are derived from a common prepropeptide [1, 2]. They exert biological functions by two 7-pass transmembrane receptors: orexin receptors types 1 and 2 (OX1R and OX2R) . Orexins are not only restricted to the hypothalamus, but are also detected in peripheral tissues including adipose tissue, the endocrine cells of the gut, adrenal gland testis, and the pancreas [4C8]. They exert biological functions that are involved in food intake, sleep-wake behaviors, arousal, energy balance, and energy expenditure [1, 2, 9, 10]. OXA can promote pancreatic hormone secretion and reduce blood glucose levels [11, 12]. OXA and OXB have been reported with apoptosis [13, 14] Neridronate and antiapoptotic [15, 16] function. OXA may act as a regulatory peptide taking part in both cell proliferation and apoptosis. The AKT serine/threonine kinase (a.k.a protein kinase B) has been considered a critical signaling molecule within eukaryotic cells. This kinase plays an important role in a variety of physiological and pathophysiological processes in different organs Rabbit Polyclonal to CEP76 systems, such as protein synthesis and transcription, angiogenesis, glycogen synthesis, and cell growth and survival . Specifically, the AKT signaling pathway plays a role in regulating islet mass. Previous studies have shown that AKT-null mice have hyperglycemia and loss of 0. 05 was considered to be statistically significant. 3. Results 3.1. Detection of OX1R Expression in INS-1 Cells Real-time PCR assays demonstrated that OX1R mRNA was endogenously expressed in INS-1 cells (Figure 1(a)). However, OX2R mRNA was not detectable under the same conditions (data not shown). OXA (10?10?M, 10?8?M, and 10?6?M) induced a significant increase of OX1R mRNA and protein levels in a dose-dependent manner (Figures 1(a) and 1(b)). Stimulation by 10?6?M OXA increased OX1R mRNA and protein 5.0-fold and 2.6-fold over basal levels, respectively ( 0.05). However, OXA treatment failed to stimulate OX1R protein expression in the presence of 10?6?M SB334867, a high-affinity OX1R-specific antagonist (Figure 1(b)). Open in a separate window 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 (* 0.05). 3.2. Effects of OXA on Proliferation and Viability of INS-1 Cells To determine the effects of OXA on cell viability and proliferation, INS-1 cells were stimulated with various concentrations of OXA (0?M, 10?10?M, 10?8?M, and 10?6?M) or 10?6?M OXA along with 10?6?M OX1R antagonist SB334867. The promoting effect of OXA on cell proliferation occurred in a concentration-dependent manner (Figure 2). Concentrations of 10?10, 10?8, and 10?6?M of OXA led to a 0.4-fold, 0.6-fold, and 0.8-fold increase, respectively, in cell proliferation. In cell viability, 10?8?M OXA and 10?6?M OXA caused a significant increase compared to the control. This effect was blocked by SB334867 (10?6?M) (Figure 2). Open in a separate window 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 Neridronate 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 (* 0.05). 3.3. OXA Protects INS-1 Cells from Apoptosis OXA treatment (10?10?M, 10?8?M, and 10?6?M) resulted in a decrease of the apoptotic index as measured by Annexin V/PI assays. Concentrations of 10?10?M, 10?8?M, and 10?6?M OXA led to a significant decrease in the rate of apoptosis in INS-1 cells compared to the control ( 0.05) (Figure 3), but it failed to protect cells against apoptosis in the presence of 10?6?M SB334867 (Figure 3). Open in.
- Wash the cells once briefly with PBS, then incubate for 15 minutes in blocking solution made up of 4% normal goat serum and 0
- When gated about viable immune cells in the tumor, the annexin V+ single-positive cells are distinct from caspase-3/7+ or apoptotic cells (Figure 1B)
- Miller 1995b)
- Each dot represents one patient, and bars indicate mean with SEM