Diacylglycerol kinase δ functions as a proliferation suppressor in pancreatic ß-cells.

Although an aberrant reduction in pancreatic ß-cell mass contributes to the pathogenesis of diabetes, the mechanism underlying the regulation of ß-cell mass is poorly understood. Here, we show that diacylglycerol kinase δ (DGKδ) is a key enzyme in the regulation of ß-cell mass. DGKδ expression was detected in the nucleus of ß-cells. We developed ß-cell-specific DGKδ knockout (ßDGKδ KO) mice, which showed lower blood glucose, higher plasma insulin levels, and better glucose tolerance compared to control mice. Moreover, an increased number of small islets and Ki-67-positive islet cells, as well as elevated cyclin B1 expression in the islets, were detected in the pancreas of ßDGKδ KO mice. DGKδ knockdown in the ß-cell line MIN6 induced significant increases in bromodeoxyuridine (BrdU) incorporation and cyclin B1 expression. Finally, we confirmed that streptozotocin-induced hyperglycemia and ß-cell loss were alleviated in ßDGKδ KO mice. Thus, suppressing the expression or enzymatic activity of DGKδ that functions as a suppressor of ß-cell proliferation could be a novel therapeutic approach to increase ß-cell mass for the treatment of diabetes.

Obligatory Role of Early Ca(2+) Responses in H2O2-Induced ß-Cell Apoptosis.

Our previous study using apoptosis analysis suggested that Ca(2+) release through inositol 1,4,5-trisphosphate (IP3) receptors and the subsequent Ca(2+) influx through store-operated channels (SOCs) constitute a triggering signal for H2O2-induced ß-cell apoptosis. In the present study, we further examined the obligatory role of early Ca(2+) responses in ß-cell apoptosis induction. H2O2 induced elevation of the cytosolic Ca(2+) concentration ([Ca(2+)]c) consisting of two phases: an initial transient [Ca(2+)]c elevation within 30 min and a slowly developing one thereafter. The first phase was almost abolished by 2-aminoethoxydiphenylborate (2-APB), which blocks IP3 receptors and cation channels including SOCs, while the second phase was only partially inhibited by 2-APB. The inhibition by 2-APB of the second phase was not observed when 2-APB was added 30 min after the treatment with H2O2. 2-APB also largely inhibited elevation of the mitochondrial Ca(2+) concentration ([Ca(2+)]m) induced by H2O2 when 2-APB was applied simultaneously with H2O2, but not when applied 30 min after H2O2 application. In addition, 2-APB inhibited the release of mitochondrial cytochrome c to the cytosol induced by H2O2 when 2-APB was applied simultaneously with H2O2 but not 30 min post-treatment. H2O2-induced [Ca(2+)]m elevation and cell death were not inhibited by Ru360, an inhibitor of the mitochondrial calcium uniporter (MCU). These results suggest that the H2O2-induced initial [Ca(2+)]c elevation, occurring within 30 min and mediated by Ca(2+) release through IP3 receptors and subsequent Ca(2+) influx through SOCs, leads to [Ca(2+)]m elevation, possibly through a mechanism independent of MCU, thereby inducing cytochrome c release and consequent apoptosis.

A transient disruption of fibroblastic transcriptional regulatory network facilitates trans-differentiation.

Transcriptional Regulatory Networks (TRNs) coordinate multiple transcription factors (TFs) in concert to maintain tissue homeostasis and cellular function. The re-establishment of target cell TRNs has been previously implicated in direct trans-differentiation studies where the newly introduced TFs switch on a set of key regulatory factors to induce de novo expression and function. However, the extent to which TRNs in starting cell types, such as dermal fibroblasts, protect cells from undergoing cellular reprogramming remains largely unexplored. In order to identify TFs specific to maintaining the fibroblast state, we performed systematic knockdown of 18 fibroblast-enriched TFs and analyzed differential mRNA expression against the same 18 genes, building a Matrix-RNAi. The resulting expression matrix revealed seven highly interconnected TFs. Interestingly, suppressing four out of seven TFs generated lipid droplets and induced PPARG and CEBPA expression in the presence of adipocyte-inducing medium only, while negative control knockdown cells maintained fibroblastic character in the same induction regime. Global gene expression analyses further revealed that the knockdown-induced adipocytes expressed genes associated with lipid metabolism and significantly suppressed fibroblast genes. Overall, this study reveals the critical role of the TRN in protecting cells against aberrant reprogramming, and demonstrates the vulnerability of donor cell's TRNs, offering a novel strategy to induce transgene-free trans-differentiations.