Molecular mechanism for regulation of glucose metabolism by Otx3

转录抑制因子Otx家族在大脑发育中起着至关重要的作用。我们之前发现了Otx家族的一个新成员Otx3，它在发育中的脑和成年胰腺ss细胞中表达。在本项目中，我们旨在通过阐明Otx3的生化特性和分析Otx3敲除小鼠来阐明Otx3的功能。生化实验表明，Otx3与Otx2在P3C （TAATCCGATTA）序列上形成异源二聚体，抑制Otx2介导的转激活。Otx3的156个氨基酸区（残基1-156）是其抑制因子活性所必需的，并被证明与Otx2直接相互作用。原位杂交证实了Otx3和Otx2在脑切片中的共定位。我们还利用随机寡核苷酸选择方法确定了Otx3的一致结合序列[TAATCCGATTA和TAATCC(N2-4)TAATCC]。为了阐明Otx3的生理作用，我们构建了Otx3基因敲除（KO）小鼠。KO小鼠存活正常，脑形态无明显异常，包括脑前/后轴分子标记的表达模式。有趣的是，KO小鼠在食物剥夺后表现出明显的低血糖，并且在口服葡萄糖负荷下胰岛素分泌减少，这表明KO小鼠在糖代谢调节方面存在缺陷。虽然Otx3在成人胰岛和胰腺ss细胞系MIN6中表达，但在KO胰腺灌注实验中，胰岛素对葡萄糖的分泌反应未受影响，表明KO ss细胞的胰岛素分泌功能保持正常。相比之下，KO小鼠对外源性胰岛素过敏。考虑到Otx3除胰腺ss细胞外几乎只在脑中表达，脑组织Otx3功能障碍可能导致葡萄糖稳态调节异常。

The Otx family of transcriptional repressors plays a critical role in the development of brain. We previously identified a novel member of the Otx family Otx3,which is expressed in developing brain and adult pancreatic ss-cells. In this project, we aimed at clarifying the function of Otx3 by elucidating biochemical properties of Otx3 and by analyzing Otx3 knockout mice.Biochemical experiments revealed that Otx3 formed heterodimer with Otx2 on P3C (TAATCCGATTA) sequence and repressed the Otx2-mediated transactivation. The 156 amino acid region (residue 1-156) of Otx3 is required for its repressor activity and was shown to interact directly with Otx2. Co-localization of Otx3 and Otx2 in the brain section was confirmed by in situ hybridization. We have also identified the consensus binding sequence [TAATCCGATTA and TAATCC(N2-4)TAATCC] of Otx3 using the random oligonucleotide selection method.To clarify the physiological roles of Otx3,Otx3 knockout (KO) mice were generated. KO mice are viable and lack gross abnormality in the morphology of brain including the expression patterns of molecular markers of anterior/posterior axis of the brain. Interestingly, KO mice exhibited marked hypoglycemia after food deprivation and decreased insulin secretion in response to oral glucose load, suggesting that KO mice are defective in the regulation of glucose metabolism. Although Otx3 is expressed in adult pancreatic islets and a pancreatic ss-cell line MIN6, insulin secretory response to glucose was not affected in the perfusion experiments of KO pancreata, suggesting that the insulin-secretory function of KO ss-cells remains normal. By contrast, KO mice are shown to be hypersensitive to exogenous insulin. Considering that Otx3 is expressed almost exclusively in brain except for pancreatic ss-cells, dysfunction of Otx3 in brain is suggested to cause the abnormal regulation of glucose homeostasis.