Intraislet Communication in Surgically-Altered Pancreas

描述（由申请人提供）： 初步数据表明，胰岛内存在δ-β细胞内分泌轴，其中胰岛内生长抑素抑制胰岛素分泌。负责抑制胰岛素的生长抑素受体亚型是未知的，然而，初步数据表明，SSTR 1和SSTR 5是胰岛素分泌的重要调节因子，并且存在种属差异。我们还证明了轴的重要性，在小鼠中SSTR 5和/或SSTR 1的基因消融导致胰岛素分泌、葡萄糖调节和胰岛形态的显著年龄和性别依赖性改变，并最终导致糖尿病。该竞争性更新提案的目的是证明以下假设：a）胰岛内生长抑素是通过SSTR 5的胰岛素分泌的重要抑制性调节剂，B）全身生长抑素是通过SSTR 1的胰岛素分泌的重要抑制性调节剂，以及c）通过SSTR 5和/或SSTR 1的基因消除破坏胰岛内和/或全身生长抑素的抑制作用将导致小鼠糖尿病。我们希望将小鼠的观察结果转化为人类胰岛，并确定SSTR 5和SSTR 1在人类内分泌胰腺中的作用，具体目标有三个：以确定a）胰岛内生长抑素是否抑制小鼠中通过SSTR 5的胰岛素分泌，B）通过SSTR 5的基因切除破坏胰岛内生长抑素的抑制作用是否导致老年小鼠中胰岛素分泌、葡萄糖稳态和胰岛形态的改变并最终导致糖尿病，以及c）这些改变是否是年龄和性别依赖性的。具体目标2。确定a）胰岛内生长抑素是否抑制小鼠通过SSTR 1的胰岛素分泌，B）通过SSTR 1的基因切除破坏胰岛内生长抑素的抑制作用是否导致胰岛素分泌、葡萄糖稳态和胰岛形态的改变并最终导致年轻小鼠的糖尿病，和c）这些改变是否是年龄和性别依赖性的，和d）SSTR 1/SSTR 5的双基因切除是否导致胰岛素分泌的改变，葡萄糖稳态和胰岛形态，并最终导致小鼠糖尿病。具体目标3。以确定a）胰岛内生长抑素是否通过SSTR 1和SSTR 5抑制人胰岛中的胰岛素分泌，和B）SSTR 1和SSTR 5的调节作用是否是年龄和性别依赖性的。 对于该提议，将随着时间的推移研究已经在我们实验室中建立的四个小鼠集落：1）SSTR 5 1/-小鼠，2）β细胞特异性SSTR 5-/-小鼠，3）SSTR 1-/-小鼠和4）SSTR &5-/-小鼠。将开发另外一个小鼠集落：1）β细胞特异性SSTR-/-小鼠。将使用以下技术进行体内和体外生理学研究：1）小鼠腹膜内葡萄糖耐量试验，2）分离的灌注小鼠胰腺，3）分离的小鼠胰岛培养物，和4）分离的灌注人胰腺。SSTR 1和SSTR 5对人和小鼠胰岛素分泌的作用将通过检查胰岛素对不同水平的葡萄糖、选择性SSTR 1和SSTR 5激动剂和有效的生长抑素单克隆抗体的反应来确定。免疫组织化学、Western印迹、北方印迹、南方印迹和RT-PCR将用于确定SSTR 1和SSTR 5基因去除是否改变小鼠胰岛中所选调节蛋白的表达，所述调节蛋白例如其它SSTR、PDX-1、PCNA、c-Myc和TGF-β和Smads。将使用光学显微镜研究小鼠中SSTR 1和SSTR 5基因消融后内分泌胰腺结构的变化。拟议的研究将确定SSTR 1和SSTR 5是否是人类和小鼠胰岛中胰岛素分泌的主要抑制性调节剂，以及在小鼠β细胞中消融这些受体是否会产生病理生理后果。将这些分子工程小鼠胰岛中的观察结果转化为人类胰岛将有望为调节人类胰岛素分泌的生理途径提供更深入的了解，这可能有助于糖尿病患者的诊断和治疗。

DESCRIPTION (provided by applicant): Preliminary data suggest the presence of a delta-to-beta cell endocrine axis within the islet in which intraislet somatostatin inhibits insulin secretion. The somatostatin receptor subtypes responsible for the inhibition of insulin are unknown, however, preliminary data suggest that SSTR1 and SSTR5 are important regulators of insulin secretion and that there are species differences. We have also demonstrated the importance of the axis in that gene ablation of SSTR5 and/or SSTR1 in mice results in significant age- and sex-dependent alterations in insulin secretion, glucose regulation and islet morphology, and ultimately in diabetes. The purpose of this competitive renewal proposal is to prove the hypotheses that a) intraislet somatostatin is an important inhibitory regulator of insulin secretion via SSTR5, b) systemic somatostatin is an important inhibitory regulator of insulin secretion via SSTR1 and c) disruption of the inhibitory effect of intraislet and/or systemic somatostatin by gene ablation of SSTR5 and/or SSTR1 will result in diabetes in mice. We hope to translate the observations in mice to human islets and determine the role of SSTR5 and SSTR1 in the human endocrine pancreas in three specific aims: Specific Aim 1. to determine whether a) intraislet somatostatin inhibits insulin secretion via SSTR5 in mice, b) disruption of the inhibitory effect of intraislet somatostatin by gene ablation of SSTR5 results in altered insulin secretion, glucose homeostasis and islet morphology and ultimately diabetes in older mice and c) the alterations are age- and sex-dependent. Specific Aim 2. to determine whether a) intraislet somatostatin inhibits insulin secretion via SSTR1 in mice, b) disruption of the inhibitory effect of intraislet somatostatin by gene ablation of SSTR1 results in altered insulin secretion, glucose homeostasis and islet morphology and ultimately diabetes in younger mice, and c) the alterations are age- and sex-dependent, and d) double gene ablation of SSTRI/SSTR5 results in altered insulin secretion, glucose homeostasis and islet morphology and ultimately diabetes in mice. Specific Aim 3. to determine whether a) intraislet somatostatin inhibits insulin secretion via SSTR1 and SSTR5 in human islets and b) the regulatory effects of SSTR1 and SSTR5 are age- and sex-dependent. For this proposal, four mouse colonies, already established in our laboratory, will be studied over time: 1) SSTR5 1/- mice, 2) beta cell-specific SSTR5 -/- mice, 3) SSTR1 -/-mice and 4) SSTRI&5 -/- mice. One additional mouse colony will be developed: 1) beta cell-specific SSTR-/- mice. In vivo and in vitro physiology studies will be performed using the following techniques: 1) intraperitoneal glucose tolerance tests in mice 2) isolated perfused mouse pancreas, 3) isolated mouse islet cultures, and 4) isolated perfused human pancreas. The role of SSTR1 and SSTR5 on human and mouse insulin secretion will be determined by examining insulin responses to varying levels of glucose, selective SSTR1 and SSTR5 agonists and to a potent somatostatin monoclonal antibody. Immunohistochemistry, Western blot, Northern blot, Southern blot and RT-PCR will be used to determine whether SSTR1 and SSTR5 gene ablation alters expression of selected regulatory proteins, such as other SSTRs, PDX-1, PCNA, c-Myc, and TGF-beta and Smads in mouse islets. Light microscopy will be used to study alterations in structure of the endocrine pancreas following SSTR1 and SSTR5 gene ablation in mice. The proposed studies will determine whether SSTR1 and SSTR5 are the predominant inhibitory regulators of insulin secretion in human and mouse islets and whether there are pathophysiologic consequences to ablating these receptors in mouse beta cells. Translation of observation in these molecularly-engineered mouse islets to human islets will hopefully provide greater insights into physiologic pathways regulating human insulin secretion, which potentially could benefit in the diagnosis and treatment of patients with diabetes.