The islet tissue plasminogen activator/plasmin system is upregulated with human islet amyloid polypeptide aggregation and protects beta cells from aggregation-induced toxicity,Diabetologia

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The islet tissue plasminogen activator/plasmin system is upregulated with human islet amyloid polypeptide aggregation and protects beta cells from aggregation-induced toxicity
Diabetologia ( IF 8.4 ) Pub Date : 2024-09-09 , DOI: 10.1007/s00125-024-06161-0
Nathalie Esser _{1,

2,

3,

4} , Meghan F Hogan _{1,

2} , Andrew T Templin _{1,

2,

5} , Rehana Akter _{1,

2} , Brendy S Fountaine ₁ , Joseph J Castillo _{1,

2} , Assam El-Osta ₆ , Lakshan Manathunga _{7,

8} , Alexander Zhyvoloup ₉ , Daniel P Raleigh _{7,

8,

9} , Sakeneh Zraika _{1,

2} , Rebecca L Hull _{1,

2} , Steven E Kahn _{1,

2}

Affiliation

Aims/hypothesis

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/interpretation

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.

Graphical Abstract

中文翻译：

胰岛组织纤溶酶原激活剂/纤溶酶系统通过人胰岛淀粉样多肽聚集上调，并保护 β 细胞免受聚集诱导的毒性

目标/假设

据报道，除了其纤溶活性外，组织纤溶酶原激活剂 (tPA)/纤溶酶系统还能裂解肽淀粉样蛋白 β，从而减轻阿尔茨海默病中的大脑淀粉样蛋白沉积。由于人胰岛淀粉样蛋白多肽 (hIAPP) 的聚集对 β 细胞具有毒性，因此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，这两种情况均在 2 型糖尿病中观察到。

方法

在来自有淀粉样蛋白倾向的 hIAPP 转基因小鼠的分离胰岛或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表达非淀粉样蛋白形成的小鼠胰岛淀粉样蛋白多肽的非转基因对照胰岛中测量 Plat（编码 tPA）的表达和纤溶酶活性。还测定了 hIAPP 处理的原代胰岛内皮细胞、骨髓源性巨噬细胞 (BMDM) 和 INS-1 细胞中的Plat表达，以确定响应 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 聚集动力学并研究纤溶酶对 hIAPP 的裂解。在使用或不使用纤溶酶的 hIAPP 处理的 INS-1 β 细胞中评估细胞活力。最后，为了证实人类样本中的发现，我们测量了患有和不患有 2 型糖尿病的捐赠者新鲜分离的胰岛中的PLAT表达。

结果

在转基因小鼠的分离胰岛中，随着淀粉样蛋白沉积的过程，胰岛Plat表达和纤溶酶活性显着增加（ p ≤0.01， n =5）；在非转基因小鼠的胰岛中未观察到这些效应，并被刚果红阻断（ p ≤0.01， n =4）。作为对 hIAPP 暴露的反应，与媒介物处理的细胞相比，BMDM 和 INS-1 细胞中Plat表达增加（ p ≤0.05， n = 4），但在胰岛内皮细胞中没有增加。在无细胞系统中，纤溶酶以剂量依赖性方式减少 hIAPP 原纤维形成，并恢复 hIAPP 诱导的 INS-1 β 细胞中细胞活力的丧失（ p ≤ 0.01， n = 5）。纤溶酶裂解单体 hIAPP，导致全长 hIAPP 丰度快速下降，并出现 hIAPP 1-11 和 12-37 片段。 hIAPP 12-37 包含关键的淀粉样蛋白生成区域，对 INS-1 细胞没有毒性。最后，与未患 2 型糖尿病的供体 ( n = 7) 的胰岛相比，患有 2 型糖尿病的供体 ( n = 4) 的胰岛中的PLAT表达显着增加 2.4 倍 ( p ≤ 0.05)。