Intraislet Communication in Surgically-Altered Pancreas

描述(由申请人提供)： 初步数据表明，胰岛内存在一个β-β细胞内分泌轴，其中胰岛内生长抑素抑制胰岛素的分泌。导致胰岛素抑制的生长抑素受体亚型尚不清楚，但初步数据表明，SSTR1和SSTR5是胰岛素分泌的重要调节因子，并且存在物种差异。我们还证明了在小鼠SSTR5和/或SSTR1基因去除导致胰岛素分泌、血糖调节和胰岛形态发生显著的年龄和性别依赖性改变，最终导致糖尿病的过程中，轴的重要性。这一竞争性更新方案的目的是证明以下假设：a)胰岛内生长抑素是通过SSTR5分泌胰岛素的重要抑制调节因子，b)全身生长抑素是通过SSTR1分泌胰岛素的重要抑制调节因子，以及c)通过SSTR5和/或SSTR1基因去除胰岛内和/或全身生长抑素的抑制作用将导致小鼠糖尿病。我们希望将在小鼠中的观察结果转化为人类胰岛，并从三个特定的目的来确定SSTR5和SSTR1在人类内分泌胰腺中的作用：具体目的1.确定a)胰岛内生长抑素是否通过SSTR5抑制小鼠的胰岛素分泌，b)通过SSTR5基因去除胰岛内生长抑素的抑制作用，导致胰岛素分泌、葡萄糖稳态和胰岛形态改变，最终导致老年小鼠糖尿病，c)这种变化是年龄和性别相关的。具体目的2.确定a)胰岛内生长抑素是否抑制小鼠通过SSTR1的胰岛素分泌，b)通过SSTR1基因去除胰岛内生长抑素的抑制作用会导致年轻小鼠胰岛素分泌、葡萄糖稳态和胰岛形态改变，并最终导致糖尿病，以及c)这种改变是年龄和性别相关的，以及d)SSTRI/SSTR5双基因切除导致胰岛素分泌改变，葡萄糖稳态和胰岛形态改变，最终导致糖尿病。具体目的3.确定a)胰岛内生长抑素是否通过SSTR1和SSTR5抑制人胰岛胰岛素的分泌，以及b)SSTR1和SSTR5的调节作用与年龄和性别有关。 根据这一建议，我们实验室已经建立的四个小鼠群体将随着时间的推移进行研究：1)SSTR5 1/-小鼠，2)β细胞特异性SSTR5-/-小鼠，3)SSTR1-/-小鼠和4)SSTRI&5-/-小鼠。另一个小鼠群体将被开发出来：1)β细胞特异性SSTR-/-小鼠。体内和体外的生理学研究将使用以下技术：1)小鼠的腹膜糖耐量试验；2)分离的灌流小鼠胰腺；3)分离的小鼠胰岛培养；4)分离的灌流人胰腺。SSTR1和SSTR5对人类和小鼠胰岛素分泌的作用将通过检测胰岛素对不同水平的葡萄糖、选择性SSTR1和SSTR5激动剂以及强大的生长抑素单抗的反应来确定。免疫组织化学、Western印迹、Northern印迹、Southern印迹和RT-PCR将被用来确定SSTR1和SSTR5基因去除是否改变了选定的调节蛋白的表达，如其他SSTR、PDX-1、增殖细胞核抗原、c-Myc、转化生长因子-β和Smads。光学显微镜将用于研究SSTR1和SSTR5基因去除后小鼠内分泌胰腺结构的变化。这项拟议的研究将确定SSTR1和SSTR5是否是人和小鼠胰岛胰岛素分泌的主要抑制调节因子，以及在小鼠β细胞中去除这些受体是否有病理生理后果。将这些分子工程小鼠胰岛中的观察转化为人类胰岛，有望为调节人类胰岛素分泌的生理途径提供更好的见解，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.