Sarkar S A, Gunter J, Bouchard R, Reusch J E-B, Wiseman A, Gill R G, Hutton J C, Pugazhenthi S
Barbara Davis Center for Childhood Diabetes and Rocky Mountain Islet Transplantation Program, Aurora, CO, USA.
Diabetologia. 2007 Aug;50(8):1649-59. doi: 10.1007/s00125-007-0707-z. Epub 2007 Jun 26.
AIMS/HYPOTHESIS: Transplantation of islets is a viable option for the treatment of diabetes. A significant proportion of islets is lost during isolation, storage and after transplantation as a result of apoptosis. cAMP response element binding protein (CREB) is an important cell survival factor. The aim of the present study was to determine whether preservation of CREB function is needed for survival of human islets.
To determine the effects of downregulation of CREB activity on beta cell apoptosis in a transplantation setting, adenoviral vectors were used to express two dominant negative mutant forms of CREB in human islets isolated from cadaveric donors. Markers of apoptosis were determined in these transduced islets under basal conditions and following treatment with growth factor.
Expression of CREB mutants in human islets resulted in significant (p < 0.001) activation of caspase-9, a key regulatory enzyme in the mitochondrial pathway of apoptosis, when compared with islets transduced with adenoviral beta galactosidase. Immunocytochemical analysis showed the activation of caspase-9 to be predominantly in beta cells. Other definitive markers of apoptosis such as parallel activation of caspase-3, accumulation of cleaved poly-(ADP-ribose) polymerase and nuclear condensation were also observed. Furthermore, the anti-apoptotic action of growth factors exendin-4 and betacellulin in human islets exposed to cytokines was partially lost when CREB function was impaired.
CONCLUSIONS/INTERPRETATION: Our findings suggest that impairment of CREB-mediated transcription could lead to loss of islets by apoptosis with potential implications in islet transplantation as well as in the mechanism of beta cell loss leading to diabetes.
目的/假设：胰岛移植是治疗糖尿病的一种可行选择。在分离、储存及移植后，相当一部分胰岛因凋亡而丢失。环磷酸腺苷反应元件结合蛋白（CREB）是一种重要的细胞存活因子。本研究的目的是确定人类胰岛的存活是否需要保留CREB功能。
为了确定在移植环境中下调CREB活性对β细胞凋亡的影响，使用腺病毒载体在从尸体供体分离的人类胰岛中表达两种显性负性突变形式的CREB。在基础条件下以及用生长因子处理后，在这些转导的胰岛中测定凋亡标志物。
与用腺病毒β半乳糖苷酶转导的胰岛相比，在人类胰岛中表达CREB突变体导致凋亡线粒体途径中的关键调节酶caspase-9显著激活（p < 0.001）。免疫细胞化学分析表明，caspase-9的激活主要发生在β细胞中。还观察到凋亡的其他确定性标志物，如caspase-3的平行激活、裂解的聚（ADP-核糖）聚合酶的积累和核浓缩。此外，当CREB功能受损时，生长因子艾塞那肽-4和β细胞ulin在暴露于细胞因子的人类胰岛中的抗凋亡作用部分丧失。
结论/解读：我们的研究结果表明，CREB介导的转录受损可能导致胰岛因凋亡而丢失，这对胰岛移植以及导致糖尿病的β细胞丢失机制具有潜在影响。
