Goping Gertrud, Pollard Harvey B, Srivastava Meera, Leapman Richard
Department of Anatomy, Physiology and Genetics, and Institute for Molecular Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
Microsc Res Tech. 2003 Aug 1;61(5):448-56. doi: 10.1002/jemt.10294.
A combination of immuno-electron microscopy and electron energy-loss spectrum-imaging was used to map the distributions of endocrine polypeptide hormones and proteins in mouse pancreatic islet of Langerhans. Tissue was analyzed from control animals and from mice that were heterozygous for the Anx7 gene, which defines a Ca2+/GTP-dependent membrane fusion and ion channel protein. The heterozygous Anx7 (+/-) mouse displays defects in IP3 receptor mediated Ca2+ signaling and insulin secretion. Therefore, information was obtained about the distributions of the hormones insulin and glucagon, as well as the proteins ANX7 and the IP3 receptor. Insulin secretion appears to be defective in the mutants. It was found from immunolabeling experiments that expression of the IP3 receptor is reduced in mutant islets compared to control islets. Subcellular distributions of sulfur and nitrogen obtained by electron energy-loss spectrum-imaging showed that the insulin concentrations of beta granules were essentially the same in control and mutant islets. By contrast, immunogold labeling of mutant islets shows more insulin immunoreactivity in the beta granules. It follows that insulin may be packaged differently in mutant islets, making antigenic determinants more available to the labeling antibody. The increased rate of insulin secretion in the hyperplastic mutant islets can be explained by compensatory increases in islet size, rather than by an increased insulin concentration in the beta cells. The results indicate that reduced ANX7 expression leads to defects in the IP3 receptor expression in the endocrine cells of the mutant mouse. Increased size of the islet or of adrenal medulla may be a compensatory mechanism for secretion defect by individual endocrine cells. Defects in IP3 receptor expression, and documented consequences of a Ca2+ signaling defect, lead to other changes in organelles such as the mitochondrial number in islet beta-cells. The effects and consequences of reduced ANX7 expression on mitochondria are evident in ultrastructural observations.
免疫电子显微镜和电子能量损失谱成像相结合的方法被用于绘制小鼠胰岛中内分泌多肽激素和蛋白质的分布图。对对照动物以及Anx7基因杂合的小鼠组织进行了分析，Anx7基因定义了一种依赖Ca2+/GTP的膜融合和离子通道蛋白。杂合的Anx7（+/-）小鼠在IP3受体介导的Ca2+信号传导和胰岛素分泌方面存在缺陷。因此，获得了有关激素胰岛素和胰高血糖素以及蛋白质ANX7和IP3受体分布的信息。突变体中的胰岛素分泌似乎存在缺陷。从免疫标记实验中发现，与对照胰岛相比，突变胰岛中IP3受体的表达降低。通过电子能量损失谱成像获得的硫和氮的亚细胞分布表明，对照胰岛和突变胰岛中β颗粒的胰岛素浓度基本相同。相比之下，突变胰岛的免疫金标记显示β颗粒中有更多的胰岛素免疫反应性。由此可见，突变胰岛中胰岛素的包装方式可能不同，使得抗原决定簇更容易与标记抗体结合。增生性突变胰岛中胰岛素分泌率的增加可以用胰岛大小的代偿性增加来解释，而不是β细胞中胰岛素浓度的增加。结果表明，ANX7表达的降低导致突变小鼠内分泌细胞中IP3受体表达的缺陷。胰岛或肾上腺髓质大小的增加可能是单个内分泌细胞分泌缺陷的一种代偿机制。IP3受体表达的缺陷以及Ca2+信号传导缺陷的记录后果，导致了细胞器的其他变化，如胰岛β细胞中的线粒体数量。在超微结构观察中，ANX7表达降低对线粒体的影响和后果是明显的。
