13C NMR (100 MHz, DMSO-d6): 165.55, 158.22, 134.98, 134.02, 131.41, 130.67, 129.93, 128.79, 128.67, 127.87, 113.67, 58.86, 55.05, 51.91, 42.13, 22.38, 21.24. the potency around the 3-(< 0.01 compared with Tm treatment alone. In addition to -cell apoptosis, ER stress also leads to -cell dysfunction, namely, the impairment of biosynthesis and secretion of insulin. First, we examined whether compound 13d reverses the Tm-suppressed mRNA levels of insulin genes. As expected, Tm treatment of INS-1 cells decreased the NPS-2143 hydrochloride mRNA levels of both insulin genes, INS1 and INS2, but this reduction was completely rescued by 13d (Figures 3A and 3B). Second, we examined whether compound 13d affects the expression of -cell transcription factors PDX1 and MafA, which control -cell identity and the expression of insulin genes.25, 26 Tm treatment decreased the levels of PDX1 and MafA mRNA expression levels in INS-1 cells, however, these decreases were completely rescued by 13d co-treatment (Figures 3C and 3D). Next, we investigated whether compound 13d re-establishes Tm-impaired glucose-stimulated insulin secretion (GSIS). As shown in Physique 3E, Tm treatment abolished the insulin secretion caused by high concentration of glucose treatment (20 mM) in INS-1 cells. Addition of 13d significantly rescued the GSIS in Tm-treated cells. Taken together, these data demonstrate that 13d restores ER stress-impaired b-cell survival and function. Open in a separate window Physique 3 Compound 13d reversed Tm-suppressed -cell dysfunction. (ACD) INS-1 cells were treated with or without Tm (0.1 g/mL) in the presence of 13d at indicated concentrations or DMSO for 24 h. The mRNA levels of INS1 (A), INS2 (B), PDX1 (C), and MafA (D), were analyzed by qRT-PCR. Relative mRNA levels were normalized against the housekeeping gene Cyclophilin A using the comparative CT method. The results are the means of 3 replicate wells and are representative of 3 impartial experiments. * < 0.05 and ** < 0.01 compared with Tm treatment alone. Bars indicate SD. (E) Insulin secretion by INS-1 cells incubated with 1.7 mM and 20 mM glucose in the presence or absence of Tm (0.1 g/mL) and 13d. Secreted insulin was measured by ELISA after 24 h Rabbit Polyclonal to DIDO1 treatment. * < 0.05. The amount of insulin secreted in response to 1 1.7 mM glucose in the absence of Tm was set to 1 1.0 and was normalized with total protein concentration. Having established that compound 13d restores ER stress-impaired b-cell survival and function, we next investigated the mechanism of action by which 13d protects -cells against ER stress. Chronic or severe ER stress activates all three branches of the UPR, PERK, IRE1a, and ATF6, leading to eventual cell death. First, we NPS-2143 hydrochloride decided the effect of 13d around the activation of the PERK pathway in -cells under ER stress. PERK activation phosphorylates eukaryotic translation initiator factor 2 (eIF2), which in turn allows for the up-regulation of activating transcription factor 4 (ATF4) and of the pro-apoptotic gene C/EBP-homologous protein (CHOP).4, 8, 11, 27 Thus, we used ATF4 and CHOP expression levels as markers of PERK pathway activation. Tm treatment of INS-1 cells significantly increased the mRNA levels of both ATF4 and CHOP, whereas co-treatment with 13d almost completely abolished the Tm-induced increase in both mRNA levels (Figures 4A and 4B). In addition, after treatment with Tm for 8 h, CHOP mRNA level NPS-2143 hydrochloride increased 7-fold compared with the control group, and co-treatment with compound 13d inhibited Tm-induced CHOP expression with an IC50 value of 0.037 M (Figure 4A). Moreover, this IC50 value was almost equal to the EC50 value of 13d for cell survival (Table 5). In addition, 13d had no effect on the mRNA levels of ATF4 and CHOP on its own (Physique 4A and 4B). Finally, consistent with the results on ATF4 and CHOP mRNA levels, 13d significantly suppressed Tm-induced increase in the protein levels of both ARF4 and CHOP (Physique 4CCE). These results indicate that 13d inhibits the activation of PERK-ATF4-CHOP pathway of the UPR under ER stress. Open in a separate window Physique 4 Compound 13d inhibits Tm-induced ATF4 and CHOP up-regulation in INS-1 cells. (A, B) INS-1 cells were NPS-2143 hydrochloride treated with or without Tm (0.1 g/mL) in the presence of 13d or DMSO for 8 h. ATF4 (A) and CHOP (B) mRNA levels were analyzed by qRT-PCR. Relative mRNA levels were normalized against the housekeeping gene Cyclophilin A using the comparative CT method. The results were expressed as the fold-increase over mRNA levels in untreated control cells and are the means of 3 replicate wells and representative of 3 impartial experiments. ** < 0.01 compared with Tm treatment alone. Bar indicates SD. (CCE) INS-1 cells were treated with or without Tm (0.1 g/mL) in the.