Han X, Sun Y, Scott S, Bleich D
Leslie and Susan Gonda (Goldschmeid) Diabetes and Genetics Research Center, Department of Diabetes, Endocrinology, and Metabolism, City of Hope National Medical Center, Duarte, California 91010, USA.
Diabetes. 2001 May;50(5):1047-55. doi: 10.2337/diabetes.50.5.1047.
In addition to inhibiting matrix metalloproteinase-2 and matrix metalloproteinase-9 activity, recent studies suggest that tissue inhibitor of metalloproteinase (TIMP)-1 may inhibit apoptosis in various cell lines. To address this question in pancreatic islets and beta-cells, we treated rat pancreatic islets and INS-1 cells with a high-dose combination of the cytokines interleukin (IL)-1beta, tumor necrosis factor-alpha, and interferon-gamma with or without the addition of TIMP-1 and TIMP-2 protein. Using flow cytometry, we quantitated DNA fragmentation to assess cellular apoptosis and confirmed these observations with DNA laddering experiments. Next, we transfected the mouse TIMP-1 gene into INS-1 cells and performed Western immunoblotting to demonstrate expression of TIMP-1 protein. We treated TIMP-1-expressing INS-1 cells with high-dose cytokines and again used flow cytometry to assess DNA fragmentation. We also evaluated the effect of TIMP-1 on IL-1beta-induced inhibition of glucose-stimulated insulin secretion (GSIS) in freshly isolated rat pancreatic islets. Finally, we evaluated the effect of TIMP-1 on inducible nitric oxide synthase (iNOS) gene expression and nuclear factor (NF)-kappaB activity in INS-1 cells stimulated with high-dose cytokines. TIMP-1 but not TIMP-2 prevented cytokine-induced apoptosis and cytokine-mediated inhibition of GSIS in rat islets and beta-cells. TIMP-1 mediated these effects by inhibiting cytokine activation of NF-kappaB, but it did not affect nitric oxide production or iNOS gene expression. Therefore, TIMP-1 may be an ideal gene to prevent cytokine-mediated beta-cell destruction and dysfunction in models of type 1 diabetes and islet transplantation rejection.
除了抑制基质金属蛋白酶-2和基质金属蛋白酶-9的活性外，最近的研究表明金属蛋白酶组织抑制剂（TIMP）-1可能抑制多种细胞系中的细胞凋亡。为了在胰岛和β细胞中探讨这个问题，我们用细胞因子白细胞介素（IL）-1β、肿瘤坏死因子-α和干扰素-γ的高剂量组合处理大鼠胰岛和INS-1细胞，添加或不添加TIMP-1和TIMP-2蛋白。使用流式细胞术，我们定量DNA片段化以评估细胞凋亡，并通过DNA梯状实验证实这些观察结果。接下来，我们将小鼠TIMP-1基因转染到INS-1细胞中，并进行蛋白质免疫印迹以证明TIMP-1蛋白的表达。我们用高剂量细胞因子处理表达TIMP-1的INS-1细胞，并再次使用流式细胞术评估DNA片段化。我们还评估了TIMP-1对新鲜分离的大鼠胰岛中IL-1β诱导的葡萄糖刺激的胰岛素分泌（GSIS）抑制的影响。最后，我们评估了TIMP-1对高剂量细胞因子刺激的INS-1细胞中诱导型一氧化氮合酶（iNOS）基因表达和核因子（NF）-κB活性的影响。TIMP-1而非TIMP-2可预防细胞因子诱导的大鼠胰岛和β细胞凋亡以及细胞因子介导的GSIS抑制。TIMP-1通过抑制细胞因子对NF-κB的激活来介导这些作用，但它不影响一氧化氮的产生或iNOS基因表达。因此，在1型糖尿病模型和胰岛移植排斥中，TIMP-1可能是预防细胞因子介导的β细胞破坏和功能障碍的理想基因。
