DIABETES MELLITUS AND ISLET AMYLOID IN ADULT FELINES

成年猫科动物的糖尿病和胰岛淀粉样蛋白

• 批准号: 3235223
• 负责人: KENNETH H JOHNSON
• 金额: $ 14.7万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-02-01 至 1992-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3235223
• 关键词: amyloid proteins; amyloidosis; blood glucose; cats; complementary DNA; diabetes mellitus; diabetes mellitus genetics; disease /disorder model; gene expression; genetic library; genetic mapping; glucagon; glucose tolerance test; glycosuria; immunochemistry; in situ hybridization; inborn metabolism disorder diagnosis; insulin; insulin sensitivity /resistance; messenger RNA; molecular pathology; monoclonal antibody; nucleic acid probes; nucleic acid sequence; pancreatic islet function; pancreatic polypeptide; peptide hormone; protein sequence; radioimmunoassay

A previously unknown putative polypeptide hormone identified as islet amyloid polypeptide (IAPP) was recently purified from islet amyloid (IA) of cats and humans with TYPE 2 diabetes mellitus (DM). In that IAPP -derived amyloid represents the most characteristic islet lesion in this from of DM in into the development of DM in cats and humans, information concerning the production and function of IAPP may provide new insights into the development of DM associated with aging. Our broad, long term objectives are to characterize the production and function of the IAPP-precursor, and to further elucidate the relationship of IAPP to DM. These objectives will be achieved through a series of experiments designed to: 1. Compare the primary chemical structure of IA-derived IAPP from multiple diabetic cats (by HPLC and amino acid sequence analysis) with that of IAPP or an IAPP precursor isolated (by affinity column separation) from pancreas or serum of normal cats. Specifically, we wish to investigate the possibility that certain structural aberrations in IAPP of diabetics are linked to transformation into amyloid fibrils; 2. Compare IAPP-precursor production in cats with and without DM (i.e., normal and normogylycemic cats with impaired glucose tolerance (IGT). Cats will be categorized by evaluations of fasting blood glucose, RIA insulin/glucagon levels and urinalysis prior to IV glucose tolerance test. LAPP-precursor production will be evaluated by immunocytochemical analysis of pancreatic B cells and by determination of serum IAPP using RIA. Pancreases will be histologically (Congo red) evaluated-red) evaluated to determine if LA deposits in nondiabetic cats are early reflections of IGT or a "prediabetic" state; 3. Elucidate the molecular biology of IAPP production in normal islets, and also in IGT and DM. LAPP cDNA will be obtained from a lambda gt11 library (obtained after isolation of islet mRNA) using oligonucleotide probes and monospecific polyclonal antibodies. IAPP expression in normal IGT and DM cats will be examined by in situ hybridization, in vitro translation, and Northern blotting; 4. Evaluate the effects of synthetic human IAPP and synthetic fragments of IAPP on the secretion of pancreatic islet hormones (i.e. insulin, glucagon, somatostatin, and pancreatic polypeptide) using direct perfusion of rat and cat pancreas. In addition, we will evaluate rat and cat pancreas and receptor binding assays;. 5. Chemically characterize IA from degus (Octodon degus), employing extraction and purification methods used by us for cat and human IA. Amyloid samples will be purified by size exclusion and revered phase HPLC for subsequent amino acid sequencing.

一种以前未知的推定多肽激素， 胰岛淀粉样多肽（IAPP）是最近从胰岛中纯化得到的 淀粉样蛋白（IA）的猫和人与2型糖尿病（DM）。 因为IAPP衍生的淀粉样蛋白代表了 胰岛病变在此从糖尿病的发生发展到糖尿病的发生 猫和人类，关于生产和功能的信息 IAPP的研究可能为DM的发展提供新的见解 与衰老有关。 我们广泛的长期目标是 表征IAPP前体的产生和功能，以及 进一步阐明IAPP与DM的关系。 这些 目标将通过一系列实验来实现 旨在： 1. 将IA衍生的IAPP的一级化学结构与 多发性糖尿病猫（通过HPLC和氨基酸序列分析） 与分离的IAPP或IAPP前体的活性（通过亲和柱 分离）从正常猫的胰腺或血清中分离。 具体地说， 我们希望调查某些结构性的 糖尿病患者IAPP的畸变与转化为 淀粉样纤维; 2. 比较有和没有DM的猫的IAPP前体产生 (i.e.,正常和糖基化正常的糖受损猫 耐受性（IGT）。 猫将根据以下评价进行分类： 空腹血糖、RIA胰岛素/胰高血糖素水平和尿分析 在IV葡萄糖耐量试验之前。 LAP-前体生产将 通过胰腺B细胞的免疫细胞化学分析进行评价 用放射免疫法测定血清IAPP。 胰腺将 组织学（刚果红）评价-红色）评价以确定 如果非糖尿病猫的LA沉积是IGT的早期反映， “糖尿病前期”状态; 3. 阐明正常人IAPP产生的分子生物学 胰岛，以及IGT和DM。LAPP cDNA将从 λ gt 11文库（分离胰岛mRNA后获得），使用 寡核苷酸探针和单特异性多克隆抗体。 将通过以下方法检测正常IGT和DM猫中的IAPP表达： 原位杂交、体外翻译和北方印迹; 4. 评价合成人IAPP和合成人IAPP的作用。 IAPP片段对胰岛激素分泌的影响 (i.e.胰岛素、胰高血糖素、生长抑素和胰多肽） 使用大鼠和猫胰腺的直接灌注。 另外我们 将评价大鼠和猫胰腺和受体结合试验;。 5. 对来自degus的IA进行化学表征， 使用我们用于猫的提取和纯化方法， 和人类IA。 淀粉样蛋白样本将通过分子排阻法进行纯化 和反相HPLC用于随后的氨基酸测序。