| Pancreatic isletβcells are unique cell clusters which secrete insulin to maintain the plasma glucose levels in a strict physiological range. Diabetes occurs when eitherβcell function is impaired orβcell mass reduces. Transcription factor FoxO1 is a key regulator of insulin signaling pathway. A series of FoxO1 studies in pancreas indicate that FoxO1 plays important roles in pancreaticβcell differentiation, proliferation, apoptosis, insulin secretion and stress resistance. Different effects of FoxO1 exhibit in various situations, suggesting that distinct downstream target genes have been activated by FoxO1. However, little has been known about what target genes mediating the regulation of FoxO1 in islet P cells. The previous study in our lab on the model of palmitate-inducedβcell defect has made some effort on answering this question by using high-flux chip screen combined with ChIP-DSL technique. The chip result showed that, in palmitate-induced pancreatic isletβcell defect, the binding of FoxO1 to multiple genes'promoter has changed. These genes could be downstream targets of FoxO1. Based on this study and focusing on the regulation of isletβcell apoptosis, we selected genes whose structure and function are related to cell proliferation, apoptosis and cell cycle control to further study. Nudt6, Pdcd21 and Cdc9111 are three of them. In this study, we found that the expressions of Nudt6 and Pdcd21 were dose-and time-dependent increased under palmitate treatment, while Cdc9111 had no change. The luciferase assay showed that the activity of Nudt6 and Pdcd21 promoter was time-dependent increased, which could be reason for the elevation of their mRNA levels. Using both chromatin immunoprecipitation (ChIP) technique and EMSA assay, combinations of FoxO1 both to Nudt6 and Pdcd21 promoter have been confirmed, which could be further enhanced after palmitate treatment. Overexpression of FoxO1 induced significant increase in both Nudt6 and Pdcd21 promoter activity, leading to the elevation of their mRNA levels. When FoxO1 expression was knocked down by small interference RNA fragment, the basal expressions of both Nudt6 and Pdcd21 are correspondingly decreased. Moreover, the elevation of Nudt6 and Pdcd21 mRNA levels stimulated by palmitate were also reversed after interference on FoxO1. Together, these results indicated that both Nudt6 and Pdcd21 were target genes of FoxO1, and their increased expressions stimulated by palmitate were mediated by FoxO1. The roles of Nudt6 and Pdcd21 in palmitate-inducedβcell apoptosis were next evaluated. We found that overexpression of Pdcd21 could further increase the percentage of apoptotic cells after palmitate stimulation, while interference of Pdcd21 partially reversed palmitate-induced apoptosis together with activated Caspase-3, indicating that activation of Caspase-3 may take part in this process. However, no matter overexpression or knockdown of Nudt6, little effect was observed on palmitate-inducedβcell apoptosis.In the present study, the binding of FoxO1 to Nudt6 or Pdcd21 gene promoter has been confirmed for the first time, identifying that both Nudt6 and Pdcd21 are FoxO1 target genes. FoxO1 mediated palmitate-induced elevation of Nudt6 and Pdcd21 expression, and increased Pdcd21 level contributed to palmitate-induced pancreatic isletβcell apoptosis, which may accomplish through activation of Caspase-3. Our findings make a further supplement to the mechanisms underlying palmitate-inducedβcell apoptosis mediated by FoxO1, which could be helpful to clarify the molecular mechanisms regulating isletβcell mass in type 2 diabetes, providing potential new clues for the prevention and therapy of diabetes. |
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