The aim of the present study was to evaluate the effects of palmitoleate on insulin secretion and insulin mRNA levels, and to investigate the transcriptional regulation of insulin. INS‑1 rat insulinoma cells were treated with palmitoleate in the presence of high glucose, and the amount of secreted insulin was measured via radioimmunoassay. Reverse transcription-quantitative polymerase chain reaction was performed to evaluate the mRNA levels of insulin and pancreatic and duodenal homeobox 1 (PDX1) under palmitoleate treatment. The levels of PDX1, peroxisome proliferator‑activated receptor gamma (PPARG), extracellular signal‑regulated kinase (ERK)1/2 and phosphorylated ERK1/2 were measured using western blot analysis. Low concentrations of palmitoleate significantly induced insulin secretion (P=0.024), whereas the mRNA levels of insulin and PDX1 were markedly reduced. However, the inhibitory effects were reversed with the addition of U0126, suggesting that the ERK1/2‑mediated pathway may be the underlying mechanism responsible for palmitoleate-induced downregulation of insulin mRNA. Exposure of INS‑1 cells to high glucose significantly increased the phosphorylation of ERK1/2 (P=0.039), which was further enhanced by palmitoleate (P=0.025). Exposure of INS‑1 cells to high glucose significantly decreased PPARG (P=0.001), which was further decreased by the addition of palmitoleate. U0126 was able to reverse the palmitoleate‑induced effects. In conclusion, the present study suggested that palmitoleate may induce insulin secretion and inhibit insulin mRNA expression in pancreatic β-cells.

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