A Novel Tool to Understand Insulin Production and Failure in Pancreatic beta-Cell

一种了解胰腺β细胞胰岛素产生和衰竭的新工具

• 批准号: 7990730
• 负责人: ISRAEL HODISH
• 金额: $ 9.99万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-20 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7990730
• 关键词: Age; Alleles; Animals; Antibodies; Award; Beta Cell; Body Weight; Breeding; C-Peptide; C57BL/6 Mouse; Cell Line; Cell Proliferation; Cells; Chimera organism; Complementary DNA; Confocal Microscopy; Coupled; Cryoultramicrotomy; Cysteine; Development; Diabetes Mellitus; Disease Progression; Disulfides; Dominant-Negative Mutation; Endoplasmic Reticulum; Enzyme-Linked Immunosorbent Assay; Exhibits; Exocytosis; Failure; Female; Fluorescence; Fluorescence Microscopy; Frozen Sections; Functional disorder; Future; Genes; Glass; Glucose; Grant; Green Fluorescent Proteins; Heterozygote; Human; Individual; Insulin; Insulin Antibodies; Islets of Langerhans; Knock-out; Label; Lead; Life; Measures; Modeling; Molecular; Monitor; Mus; Mutation; Neonatal; Non-Insulin-Dependent Diabetes Mellitus; Operative Surgical Procedures; Pancreas; Pathway interactions; Patients; Peptides; Phase; Phenotype; Point Mutation; Population; Prevalence; Process; Production; Proinsulin; Proteins; Radioimmunoassay; Relative (related person); Rodent; Secretory Vesicles; Staging; Structure of beta Cell of islet; Sulfhydryl Compounds; Techniques; Therapeutic Intervention; Tissues; Transgenes; Transgenic Animals; Transgenic Mice; Ursidae Family; Work; diabetic; disulfide bond; fasting blood glucose level; fluorescence imaging; in vivo; insulin secretion; interest; islet; leptin receptor; male; mutant; neonate; novel; novel strategies; prevent; promoter; protein complex; protein expression; response; secretion process; sex; tool; trafficking

As the prevalence of Diabetes Mellitus increases, the need for recognizing the major cellular and molecular processes that underlie progression of the disease becomes more urgent. It is becoming increasingly clear that in patients with type 2 diabetes, P-cells are unable to fully compensate to maintain euglycemia overtime. 3-cell deficiency is preceded by a period in which p-cells are still viable but exhibit defective insulin storage/secretion. The endoplasmic reticulim (ER) becomes packed with proinsulin while production of normal insulin declines. This "golden period" before p-cell demise is of particular interest since it may highlight reversible processes that could be attacked both scientifically and therapeutically. A currently favored hypothesis is that proinsulin misfolding exacerbates 3-cell dysfunction, ultimately decreasing pancreatic insulin content and 3-cell mass. As in the Akita mouse, point mutations in proinsulin that alter its folding result in persistent congenital diabetes despite abundant nonmutant proinsulin available from wild-type alleles (autosomal dominant inheritance). In the K99 years, I have developed a novel approach to study insulin content and 3-cell mass in live animals, as well as to explore potential interactions between normal and misfolded proinsulin. Human proinsulin labeled with mid-region green fluorescent protein (GFP) can mimic the normal folding, trafficking, processing, and secretion of the native proinsulin in cell lines as well as in a transgenic mouse line called hProins-CpepGFP. In the ROO grant I am working on a new model in which the Akita mutation is introduced directly into the hProins-CpepGFP transgene (in cis) to study the fate of the misfolded proinsulin. I have also developed a new transgenic mouse line bearing GFP tagged Akita proinsulin that also bears an additional point mutation eliminating the partner (B7) free thiol group to be monitored for development of dominant negative diabetes. I plan to breed the hProinsC(A7)Y-CpepGFP line against Leptin receptor deficient db-/- mice in order to examine pathogenic synergies between proinsulin misfolding and increased insulin secretory demand. These studies will explore potentially reversible pathways leading to type 2 diabetes.

随着糖尿病患病率的增加，认识疾病进展的主要细胞和分子过程的需要变得更加紧迫。越来越清楚的是，在 2 型糖尿病患者中，P 细胞无法完全代偿以长时间维持正常血糖。 3 细胞缺陷之前有一段时期，其中 p 细胞仍然存活，但表现出胰岛素储存/分泌缺陷。内质网 (ER) 充满胰岛素原，而正常胰岛素的产生量下降。 p细胞死亡之前的这个“黄金时期”特别令人感兴趣，因为它可能强调可逆过程，而这些过程可能在科学上和治疗上受到攻击。目前流行的假设是胰岛素原错误折叠会加剧 3 细胞功能障碍，最终降低胰腺胰岛素含量和 3 细胞质量。与秋田小鼠一样，尽管野生型等位基因（常染色体显性遗传）中存在丰富的非突变胰岛素原，但胰岛素原的点突变改变了其折叠，导致持续性先天性糖尿病。在 K99 年，我开发了一种新方法来研究活体动物的胰岛素含量和 3 细胞质量，并探索正常胰岛素原和错误折叠胰岛素原之间的潜在相互作用。用中区绿色荧光蛋白 (GFP) 标记的人胰岛素原可以模拟细胞系以及称为 hProins-CpepGFP 的转基因小鼠系中天然胰岛素原的正常折叠、运输、加工和分泌。在 ROO 资助中，我正在研究一种新模型，其中将秋田突变直接引入 hProins-CpepGFP 转基因（顺式）中，以研究错误折叠的胰岛素原的命运。我还开发了一种新的转基因小鼠品系，带有 GFP 标记的秋田岛胰岛素原，该小鼠品系还带有额外的点突变，消除了伴侣 (B7) 游离硫醇基团，以监测显性阴性糖尿病的发展。我计划针对瘦素受体缺陷的 db-/- 小鼠培育 hProinsC(A7)Y-CpepGFP 系，以检查胰岛素原错误折叠和胰岛素分泌需求增加之间的致病协同作用。这些研究将探索导致 2 型糖尿病的潜在可逆途径。