PDF(Pubmed)
Abstract：
OBJECTIVE: 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.
METHODS: Here, we dissected the contribution of RIPK1 scaffold versus kinase functions to β-cell death regulation using mice lacking RIPK1 specifically in β-cells (Ripk1β-KO mice) or expressing a kinase-dead version of RIPK1 (Ripk1D138N mice), respectively. These mice were challenged with streptozotocin, a model of autoimmune diabetes. Moreover, Ripk1β-KO 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.
RESULTS: Inhibition of RIPK1 kinase activity (Ripk1D138N 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.
CONCLUSIONS: 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.
METHODS: Here, we dissected the contribution of RIPK1 scaffold versus kinase functions to β-cell death regulation using mice lacking RIPK1 specifically in β-cells (Ripk1β-KO mice) or expressing a kinase-dead version of RIPK1 (Ripk1D138N mice), respectively. These mice were challenged with streptozotocin, a model of autoimmune diabetes. Moreover, Ripk1β-KO 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.
RESULTS: Inhibition of RIPK1 kinase activity (Ripk1D138N 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.
CONCLUSIONS: 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.
摘要：
目的：受体相互作用蛋白激酶1（RIPK1）协调肿瘤坏死因子（TNF）和其他细胞因子在细胞存活和细胞死亡之间的决定。而RIPK1的支架功能对于防止TNF诱导的细胞凋亡和坏死性凋亡至关重要，它的激酶活性是细胞坏死所必需的，部分是细胞凋亡所必需的。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，TNF诱导β细胞死亡的机制尚不清楚。
方法：这里，我们使用缺乏RIPK1的小鼠（Ripk1β-KO小鼠）或表达激酶死亡版本的RIPK1（Ripk1D138N小鼠），分析了RIPK1支架与激酶功能对β细胞死亡调节的贡献，分别。这些小鼠被链脲佐菌素攻击，自身免疫性糖尿病模型.此外，用高脂肪饮食进一步攻击Ripk1β-KO小鼠以诱导高血糖。对于机械学研究，胰岛接受了各种杀伤剂和致敏剂。
结果：抑制RIPK1激酶活性（Ripk1D138N小鼠）不影响1型糖尿病模型中高血糖的发作和进展。此外，β细胞中RIPK1表达缺失不影响基础条件下的血糖正常或糖尿病挑战下的高血糖.离体,在没有RIPK1的情况下，原代胰岛对TNF诱导的凋亡和坏死不敏感。有趣的是，我们发现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(Caspase-8)和坏死(RIPK3)成分.环己酰亚胺处理,这导致了cFLIP水平的降低，使原代胰岛对TNF诱导的细胞死亡敏感，而TNF诱导的细胞死亡被胱天蛋白酶抑制完全阻断。
结论：与许多其他细胞类型不同（例如，上皮，和免疫），在生理条件或糖尿病挑战下，RIPK1对于β细胞中的细胞死亡调节不是必需的。此外，体内和体外证据表明，胰腺β细胞不会发生坏死，而主要是响应TNF的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,该模式独立于RIPK1,并且可能高度依赖于cFLIP.
方法：这里，我们使用缺乏RIPK1的小鼠（Ripk1β-KO小鼠）或表达激酶死亡版本的RIPK1（Ripk1D138N小鼠），分析了RIPK1支架与激酶功能对β细胞死亡调节的贡献，分别。这些小鼠被链脲佐菌素攻击，自身免疫性糖尿病模型.此外，用高脂肪饮食进一步攻击Ripk1β-KO小鼠以诱导高血糖。对于机械学研究，胰岛接受了各种杀伤剂和致敏剂。
结果：抑制RIPK1激酶活性（Ripk1D138N小鼠）不影响1型糖尿病模型中高血糖的发作和进展。此外，β细胞中RIPK1表达缺失不影响基础条件下的血糖正常或糖尿病挑战下的高血糖.离体,在没有RIPK1的情况下，原代胰岛对TNF诱导的凋亡和坏死不敏感。有趣的是，我们发现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(Caspase-8)和坏死(RIPK3)成分.环己酰亚胺处理,这导致了cFLIP水平的降低，使原代胰岛对TNF诱导的细胞死亡敏感，而TNF诱导的细胞死亡被胱天蛋白酶抑制完全阻断。
结论：与许多其他细胞类型不同（例如，上皮，和免疫），在生理条件或糖尿病挑战下，RIPK1对于β细胞中的细胞死亡调节不是必需的。此外，体内和体外证据表明，胰腺β细胞不会发生坏死，而主要是响应TNF的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,该模式独立于RIPK1,并且可能高度依赖于cFLIP.
