目的:除了纤溶活性外,组织纤溶酶原激活剂(tPA)/纤溶酶系统已被报道切割肽淀粉样β,减轻阿尔茨海默病中脑淀粉样蛋白沉积。由于人胰岛淀粉样多肽(hIAPP)的聚集对β细胞具有毒性,我们试图确定纤溶系统的激活是否也可以减少胰岛淀粉样蛋白沉积及其细胞毒性作用,这两者都在2型糖尿病中观察到。
方法:在不存在或存在淀粉样蛋白抑制剂刚果红的情况下培养的来自淀粉样蛋白易感hIAPP转基因小鼠的分离胰岛或非转基因对照胰岛中测量Plat(编码tPA)的表达和纤溶酶活性。在hIAPP处理的原代胰岛内皮细胞中也测定了Plat表达,骨髓源性巨噬细胞(BMDM)和INS-1细胞,以确定响应于hIAPP聚集而产生tPA的胰岛细胞类型。使用无细胞硫黄素-T测定和MS分别监测hIAPP聚集动力学并研究hIAPP的纤溶酶裂解。在用有或没有纤溶酶的hIAPP处理的INS-1β细胞中评估细胞活力。最后,为了证实人体样本中的发现,在来自有和没有2型糖尿病的供体的新鲜分离的胰岛中测量PLAT表达。
结果:在来自转基因小鼠的分离胰岛中,胰岛Plat表达和纤溶酶活性随着淀粉样蛋白沉积的过程而显着增加(p≤0.01,n=5);在非转基因小鼠的胰岛中未观察到这些作用,并且被刚果红阻断(p≤0.01,n=4)。作为对hIAPP曝光的回应,与载体处理的细胞相比,BMDM和INS-1细胞中的Plat表达增加(p≤0.05,n=4),但不在胰岛内皮细胞中。纤溶酶在无细胞系统中以剂量依赖性方式减少hIAPP原纤维形成,并恢复了hIAPP诱导的INS-1β细胞中细胞活力的丧失(p≤0.01,n=5)。纤溶酶裂解的单体hIAPP,诱导全长hIAPP的丰度迅速降低以及hIAPP1-11和12-37片段的出现。hIAPP12-37,其中包含关键的淀粉样区域,对INS-1细胞没有毒性。最后,来自2型糖尿病供体(n=4)的胰岛与来自无2型糖尿病供体(n=7)的胰岛中PLAT表达显着增加2.4倍(p≤0.05)。
结论:在具有hIAPP聚集的胰岛中,纤溶系统上调。纤溶酶快速降解hIAPP,限制其聚集成淀粉样蛋白,从而保护β细胞免受hIAPP诱导的毒性。因此,增加胰岛纤溶酶活性可能是限制2型糖尿病患者β细胞丢失的策略.
OBJECTIVE: Apart from its fibrinolytic activity, the tissue plasminogen activator (tPA)/plasmin system has been reported to cleave the peptide amyloid beta, attenuating brain amyloid deposition in Alzheimer\'s disease. As aggregation of human islet amyloid polypeptide (hIAPP) is toxic to beta cells, we sought to determine whether activation of the fibrinolytic system can also reduce islet amyloid deposition and its cytotoxic effects, which are both observed in type 2 diabetes.
METHODS: The expression of Plat (encoding tPA) and plasmin activity were measured in isolated islets from amyloid-prone hIAPP transgenic mice or non-transgenic control islets expressing non-amyloidogenic mouse islet amyloid polypeptide cultured in the absence or presence of the amyloid inhibitor Congo Red. Plat expression was also determined in hIAPP-treated primary islet endothelial cells, bone marrow-derived macrophages (BMDM) and INS-1 cells, in order to determine the islet cell type(s) producing tPA in response to hIAPP aggregation. Cell-free thioflavin-T assays and MS were used to respectively monitor hIAPP aggregation kinetics and investigate plasmin cleavage of hIAPP. Cell viability was assessed in INS-1 beta cells treated with hIAPP with or without plasmin. Finally, to confirm the findings in human samples, PLAT expression was measured in freshly isolated islets from donors with and without type 2 diabetes.
RESULTS: In isolated islets from transgenic mice, islet Plat expression and plasmin activity increased significantly with the process of amyloid deposition (p≤0.01, n=5); these effects were not observed in islets from non-transgenic mice and were blocked by Congo Red (p≤0.01, n=4). In response to hIAPP exposure, Plat expression increased in BMDM and INS-1 cells vs vehicle-treated cells (p≤0.05, n=4), but not in islet endothelial cells. Plasmin reduced hIAPP fibril formation in a dose-dependent manner in a cell-free system, and restored hIAPP-induced loss of cell viability in INS-1 beta cells (p≤0.01, n=5). Plasmin cleaved monomeric hIAPP, inducing a rapid decrease in the abundance of full-length hIAPP and the appearance of hIAPP 1-11 and 12-37 fragments. hIAPP 12-37, which contains the critical amyloidogenic region, was not toxic to INS-1 cells. Finally, PLAT expression was significantly increased by 2.4-fold in islets from donors with type 2 diabetes (n=4) vs islets from donors without type 2 diabetes (n=7) (p≤0.05).
CONCLUSIONS: The fibrinolytic system is upregulated in islets with hIAPP aggregation. Plasmin rapidly degrades hIAPP, limiting its aggregation into amyloid and thus protecting beta cells from hIAPP-induced toxicity. Thus, increasing islet plasmin activity might be a strategy to limit beta cell loss in type 2 diabetes.