Department of Translational Genomics, Faculty of Medicine, University of Cologne, Cologne, Germany; Centre for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany.
Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany; Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany.
Mol Metab. 2024 Sep;87:101988. doi: 10.1016/j.molmet.2024.101988. Epub 2024 Jul 14.
Receptor-interacting protein kinase 1 (RIPK1) orchestrates the decision between cell survival and cell death in response to tumor necrosis factor (TNF) and other cytokines. Whereas the scaffolding function of RIPK1 is crucial to prevent TNF-induced apoptosis and necroptosis, its kinase activity is required for necroptosis and partially for apoptosis. Although TNF is a proinflammatory cytokine associated with β-cell loss in diabetes, the mechanism by which TNF induces β-cell demise remains unclear.
Here, we dissected the contribution of RIPK1 scaffold versus kinase functions to β-cell death regulation using mice lacking RIPK1 specifically in β-cells (Ripk1 mice) or expressing a kinase-dead version of RIPK1 (Ripk1 mice), respectively. These mice were challenged with streptozotocin, a model of autoimmune diabetes. Moreover, Ripk1 mice were further challenged with a high-fat diet to induce hyperglycemia. For mechanistic studies, pancreatic islets were subjected to various killing and sensitising agents.
Inhibition of RIPK1 kinase activity (Ripk1 mice) did not affect the onset and progression of hyperglycemia in a type 1 diabetes model. Moreover, the absence of RIPK1 expression in β-cells did not affect normoglycemia under basal conditions or hyperglycemia under diabetic challenges. Ex vivo, primary pancreatic islets are not sensitised to TNF-induced apoptosis and necroptosis in the absence of RIPK1. Intriguingly, we found that pancreatic islets display high levels of the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) and low levels of apoptosis (Caspase-8) and necroptosis (RIPK3) components. Cycloheximide treatment, which led to a reduction in cFLIP levels, rendered primary islets sensitive to TNF-induced cell death which was fully blocked by caspase inhibition.
Unlike in many other cell types (e.g., epithelial, and immune), RIPK1 is not required for cell death regulation in β-cells under physiological conditions or diabetic challenges. Moreover, in vivo and in vitro evidence suggest that pancreatic β-cells do not undergo necroptosis but mainly caspase-dependent death in response to TNF. Last, our results show that β-cells have a distinct mode of regulation of TNF-cytotoxicity that is independent of RIPK1 and that may be highly dependent on cFLIP.
受体相互作用蛋白激酶 1（RIPK1）在响应肿瘤坏死因子（TNF）和其他细胞因子时协调细胞存活和死亡之间的决定。虽然 RIPK1 的支架功能对于防止 TNF 诱导的细胞凋亡和坏死至关重要，但它的激酶活性对于坏死和部分凋亡是必需的。尽管 TNF 是一种与糖尿病中β细胞丢失相关的促炎细胞因子，但 TNF 诱导β细胞死亡的机制仍不清楚。
在这里，我们使用分别在β细胞中特异性缺乏 RIPK1（Ripk1 小鼠）或表达激酶失活形式的 RIPK1（Ripk1 小鼠）的小鼠，分别解析了 RIPK1 支架与激酶功能对β细胞死亡调节的贡献。这些小鼠受到链脲佐菌素的挑战，这是一种自身免疫性糖尿病模型。此外，Ripk1 小鼠进一步受到高脂肪饮食的挑战，以诱导高血糖。对于机制研究，将胰岛暴露于各种杀伤和敏化剂中。
抑制 RIPK1 激酶活性（Ripk1 小鼠）并不影响 1 型糖尿病模型中高血糖的发生和进展。此外，β细胞中 RIPK1 的缺失并不影响基础条件下的正常血糖或糖尿病挑战下的高血糖。在体外，缺乏 RIPK1 的情况下，原代胰腺胰岛对 TNF 诱导的细胞凋亡和坏死并不敏感。有趣的是，我们发现胰腺胰岛显示高水平的抗凋亡细胞 FLICE 抑制蛋白（cFLIP）和低水平的凋亡（半胱天冬酶-8）和坏死（RIPK3）成分。细胞松弛素 D 处理，导致 cFLIP 水平降低，使原代胰岛对 TNF 诱导的细胞死亡敏感，而 caspase 抑制可完全阻断该死亡。
与许多其他细胞类型（例如上皮和免疫细胞）不同，在生理条件或糖尿病挑战下，RIPK1 不是β细胞死亡调节所必需的。此外，体内和体外的证据表明，胰腺β细胞在响应 TNF 时不会发生坏死，而是主要发生 caspase 依赖性死亡。最后，我们的结果表明，β细胞具有独特的 TNF 细胞毒性调节模式，独立于 RIPK1，并且可能高度依赖于 cFLIP。
