Fueger Patrick T, Hernandez Angelina M, Chen Yi-Chun, Colvin E Scott
Department of Pediatrics, Indiana University School of Medicine, IN, USA. .
J Vis Exp. 2012 Jun 25(64):4080. doi: 10.3791/4080.
Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes (1-3). Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes. Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa. By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass (4-6). Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets (4,7-15). Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function (5,6,8,9,16,17).
葡萄糖稳态主要由分别从胰腺β细胞和α细胞分泌的内分泌激素胰岛素和胰高血糖素控制。功能性β细胞量由解剖学上的β细胞量以及β细胞对营养负荷作出反应的能力决定。功能性β细胞量的丧失是两种主要糖尿病类型的核心问题（1-3）。在1型糖尿病中，功能性β细胞量的下降是由自身免疫攻击导致的，而在2型糖尿病中，这种减少是由于β细胞无法适当分泌胰岛素以及多种机制导致的β细胞破坏。因此，恢复功能性β细胞量的努力对于更好地治疗糖尿病和实现潜在治愈至关重要。目前正在努力确定可用于刺激β细胞复制并增强其功能的分子途径。理想情况下，治疗靶点应能改善β细胞的生长和功能。不过，或许更重要的是确定刺激β细胞生长的策略是否会以损害β细胞功能为代价（例如某些癌基因的情况），反之亦然。通过系统地抑制或过表达分离的大鼠胰岛中靶基因的表达，可以确定增加功能性β细胞量的潜在治疗靶点（4-6）。腺病毒载体可用于在分离的大鼠胰岛中高效过表达或敲低蛋白质（4,7-15）。在此，我们介绍一种利用腺病毒转导来操纵基因表达并评估分离的大鼠胰岛中胰岛复制和β细胞功能的方法（图1）。该方法先前已被用于确定调节β细胞复制或功能的新靶点（5,6,8,9,16,17）。
