Sequential global gene expression analysis of glucose stimulated human islets

Islet transplantation has the potential to benefit patients with type I diabetes, but this cellular therapy is limited by a shortage of islets, which necessitates the collection or production of islets from alternative sources. If islets produced from stem cells are to be used for transplant therapy they should precisely replicate beta-cell function. Characterization of the unique molecular mechanisms underlying the beta-cell’s response to glucose stimulation will allow a better understanding of critical elements that the alternative cells must possess. Human islets from five donors were stimulated with glucose for various durations of time to discover the glucose-responsive pathways operating at a molecular level in islets. Compared with human hepatocytes, fibroblasts and peripheral blood mononuclear cells, human islets displayed distinct gene expression signatures. Seven hundred and forty genes were differentially expressed in islets during short-term glucose stimulation and 42 of these genes were exclusively expressed in terms of tissue specificity and exposure duration. One thousand six hundred and thirty five genes were differentially expressed in islets during long-term glucose stimulation and 148 of these genes were exclusively expressed. The expression of two important glucose-responsive genes TSC2 and ARF1 were measured by real-time PCR in mouse MIN6 beta-cell line. Insulin secretion from ARF1 knockdown MIN6 cells decreased by 1-fold compared with controls, and TSC2-knockdown MIN6 cells decreased by 2-fold. These findings may allow for better direction of the differentiation of other types of cells such as embryonic and induced pluripotent stem cell into insulin-producing cells. We performed gene expression profiling on samples of human pancreatic islets from 5 donors stimulated by 300 mg/dl glucose for 1 hr (5 biological repeats), 2 hr (3 biological repeats) and 24 hr (5 biological repeats) for a total of 13 arrays. For comparison, gene expression profiling was conducted on unstimulated pancreatic islets for 1 hr (4 biological repeats), 2 hr (5 biological repeats) and 24 hr (4 biological repeats) for a total of 13 arrays. Single samples of fibroblasts were treated for 1 hr and 24 hr and untreated for 1 hr, 2 hr and 24 hr periods for a total of 5 arrays. Hepatocytes were treated for 1 hr (1 sample), 2 hr (1 sample) and 24 hours (2 technical repeats) and untreated for 1 hr (1 sample), 2 hr (2 technical repeats) and 24 hr (2 technical repeats) for a total of 9 arrays. PBMC were treated for 1 hr (2 technical repeats), 2 hr (2 technical repeats) and 24 hr (2 technical repeats) and untreated for 1 hr (2 technical repeats), 2 hr (2 technical repeats) and 24 hrs (2 technical repeats) for a total of 12 arrays. Total RNA from PBMCs pooled from six normal donors was amplified into aRNA to serve as the reference.