目标:在2型糖尿病中,β细胞失败是由细胞质量损失引起的,主要是通过细胞凋亡,但也有简单的功能障碍(去分化,葡萄糖刺激的胰岛素分泌下降)。导致细胞凋亡和功能障碍,至少在某种程度上,通过葡萄糖毒性,其中,己糖胺生物合成途径中葡萄糖通量的增加起作用。在这项研究中,我们试图澄清是否增加己糖胺生物合成途径通量影响β细胞生理学的另一个重要方面,即β细胞-β细胞同型相互作用。
方法:我们使用INS-1E细胞和鼠胰岛。免疫荧光法检测E-cadherin和β-catenin的表达和细胞分布,免疫组织化学和蛋白质印迹。通过悬滴聚集测定法检查细胞-细胞粘附,通过分离和显微镜观察胰岛结构。
结果:E-cadherin表达没有因己糖胺生物合成途径通量的增加而改变,然而,细胞表面减少,细胞内E-钙粘蛋白的增加。此外,细胞内E-钙粘蛋白离域,至少在某种程度上,从高尔基复合体到内质网.发现β-连环蛋白与E-钙粘蛋白的再分布平行,显示从质膜到细胞质的错位。这些变化的表型结果是INS-1E聚集能力降低。最后,在离体实验中,葡萄糖胺能够改变胰岛结构并降低E-cadherin和β-catenin的表面丰度。
结论:增加的己糖胺生物合成途径通量改变了E-cadherin在INS-1E细胞和小鼠胰岛中的细胞定位,并影响细胞间粘附和胰岛形态。这些变化可能是由E-cadherin功能的改变引起的,突出了一个新的潜在目标,以抵消葡萄糖毒性对β细胞的影响。
In type 2 Diabetes, β-cell failure is caused by loss of cell mass, mostly by apoptosis, but also by simple dysfunction (dedifferentiation, decline of glucose-stimulated insulin secretion). Apoptosis and dysfunction are caused, at least in part, by glucotoxicity, in which increased flux of glucose in the hexosamine biosynthetic pathway plays a role. In this study, we sought to clarify whether increased hexosamine biosynthetic pathway flux affects another important aspect of β-cell physiology, that is β-cell-β-cell homotypic interactions.
We used INS-1E cells and murine islets. The expression and cellular distribution of E-cadherin and β-catenin was evaluated by immunofluorescence, immunohistochemistry and western blot. Cell-cell adhesion was examined by the hanging-drop aggregation assay, islet architecture by isolation and microscopic observation.
E-cadherin expression was not changed by increased hexosamine biosynthetic pathway flux, however, there was a decrease of cell surface, and an increase in intracellular E-cadherin. Moreover, intracellular E-cadherin delocalized, at least in part, from the Golgi complex to the endoplasmic reticulum. Beta-catenin was found to parallel the E-cadherin redistribution, showing a dislocation from the plasmamembrane to the cytosol. These changes had as a phenotypic consequence a decreased ability of INS-1E to aggregate. Finally, in ex vivo experiments, glucosamine was able to alter islet structure and to decrease surface abundandance of E-cadherin and β-catenin.
Increased hexosamine biosynthetic pathway flux alters E-cadherin cellular localization both in INS-1E cells and murine islets and affects cell-cell adhesion and islet morphology. These changes are likely caused by alterations of E-cadherin function, highlighting a new potential target to counteract the consequences of glucotoxicity on β-cells.