Molecular mechanism for regulation of glucose metabolism by Otx3

Otx家族转录抑制因子在脑发育过程中起着重要作用。我们以前确定了一个新的成员的OTX家族OTX 3，这是在发展中的大脑和成人胰腺SS细胞表达。本研究通过对Otx 3的生物化学性质的研究，以及对Otx 3基因敲除小鼠的分析，进一步阐明Otx 3的功能，结果发现Otx 3与Otx 2在P3 C（TAATCCGATTA）序列上形成异源二聚体，并抑制Otx 2介导的反式激活。Otx 3的156个氨基酸区域（残基1-156）是其阻遏活性所需的，并且显示出直接与Otx 2相互作用。通过原位杂交证实了Otx 3和Otx 2在脑切片中的共定位。我们还利用随机寡核苷酸选择方法鉴定了Otx 3的共有结合序列[TAATCCGATTA和TAATCC（N2-4）TAATCC]。KO小鼠存活，脑形态学无明显异常，包括脑前/后轴分子标志物的表达模式。有趣的是，KO小鼠在食物剥夺后表现出明显的低血糖，并且响应于口服葡萄糖负荷而降低胰岛素分泌，这表明KO小鼠在葡萄糖代谢的调节方面存在缺陷。虽然Otx 3在成年胰岛和胰腺ss细胞系MIN 6中表达，但在KO胰腺的灌注实验中，对葡萄糖的胰岛素分泌反应不受影响，表明KO ss细胞的胰岛素分泌功能保持正常。相比之下，KO小鼠显示对外源性胰岛素过敏。考虑到Otx 3几乎只在脑中表达，除了胰腺ss细胞，脑中Otx 3的功能障碍被认为是导致葡萄糖稳态调节异常的原因。

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.