Role of PDX1 in insulin biosynthesis and ER homeostasis

描述(由申请人提供)： 近几十年来，美国的人口数量有增无减 被诊断为糖尿病；因此，目前有2300万人患有2型糖尿病。这个 2型糖尿病的发病机制涉及胰岛素抵抗和胰岛素抵抗失败之间的复杂相互作用。 胰腺细胞代偿。细胞代偿是通过改善细胞功能和扩大细胞质量来实现的，这两者都受到关键的胰腺转录因子PDXI的调节。初步数据显示，在饮食诱导的胰岛素抵抗过程中，PDXI缺乏通过增加对内质网(ER)应激的敏感性而增加细胞凋亡(M.Sachdeva，未发表的数据，2008年)。细胞的内质网每分钟处理一百万个胰岛素分子，内质网压力与小鼠和人类的2型糖尿病有关。高通量基因表达和启动子占有率分析表明，PDXI调控几个参与维持ER动态平衡的基因，包括EROlip和Grp170。PDXI对EROlip和Grp170的调节被认为对胰岛素的生物合成和细胞生存是重要的。这一假说将在以下特定目标进行检验：1.确定DX1在胰岛素生物合成中的作用；2.确定PDXI在调节内质网稳态和p细胞存活中的作用。将用shRNA沉默的EROlip和Grp170研究小鼠胰岛素瘤细胞的胰岛素分泌。我们将分析EROlip在胰岛素二硫键形成中的作用以及Grp170作为细胞伴侣的功能。为了确定EROlip和Grp170缺乏对内质网稳态的影响，将检测ER应激标志物如Bip和CHOP，并使用Annexin V和TUNEL染色来定量细胞凋亡。EROlip和Grp170的过表达可能部分挽救了PDXI缺乏的表型，提示它们是PDXI影响胰岛素分泌和细胞存活的重要中介。 相关性：细胞不能充分分泌胰岛素，这些产生胰岛素的细胞死亡会导致糖尿病。研究PDXI调节胰岛素分泌和细胞存活的机制可能为2型糖尿病的治疗提供靶点。

DESCRIPTION (provided by applicant): In recent decades, the United States has experienced an unabated rise in the number of individuals diagnosed with diabetes; as a result 23 million people are currently living with type 2 diabetes. The pathogenesis of type 2 diabetes involves a complex interplay between insulin resistance and failure of pancreatic ¿ cell compensation. ¿ cell compensation occurs through improved ¿ cell function and ¿ cell mass expansion, which are both regulated by the critical pancreatic transcription factor PDXI. Preliminary data show that PDXI deficiency increases ¿ cell apoptosis during diet induced insulin resistance by increasing susceptibility to endoplasmic reticulum (ER) stress (M. Sachdeva, unpublished data, 2008). The ER of a ¿ cell processes one million insulin molecules per minute, and ER stress has been associated with type 2 diabetes in mice and humans. High throughput gene expression and promoter occupancy analyses suggest that PDXI regulates several genes involved in maintaining ER homeostasis, including EROlip and GRP170. PDXI regulation of EROlip and GRP170 is hypothesized to be important for insulin biosynthesis and ¿ cell survival. This hypothesis will be tested in the following specific aims: I. Determine the role of DX1 in insulin biosynthesis, II. Determine the role of PDXI in the regulation of ER homeostasis and p cell survival. Insulin secretion will be studied in mouse insulinoma cells with shRNA silenced EROlip and GRP170. The role of EROlip in insulin disulfide bond formation and the function of GRP170 as a ¿ cell chaperone will be analyzed. To determine the effects of EROlip and GRP170 deficiency on ER homeostasis, ER stress markers such as BiP and CHOP will be measured, and Annexin V and TUNEL staining will be used to quantify ¿ cell apoptosis. Overexpression of EROlip and GRP170 may partially rescue the PDXI deficiency phenotype suggesting that they are important mediators of the effects of PDXI on insulin secretion and ¿ cell survival. Relevance: Failure of ¿ cells to sufficiently secrete insulin and death of these insulin-producing cells results in diabetes. Studying the mechanism for PDXI regulation of insulin secretion and ¿ cell survival may lead to targets for treatment of type 2 diabetes.