Illumina-microarray analysis of mycophenolic acid-induced cell death in an insulin-producing cell line and primary rat islet cells: new insights into apoptotic pathways involved

Mycophenolic acid (MPA), widely used to prevent organ transplant rejection, may induce toxicity and impair function in beta-cells. Mechanisms of MPA-induced cell death have not been fully explored. In this study, we examined gene expression patterns in INS-1E cells and isolated primary rat islets following MPA treatment using the Illumina-cDNA microarray. The MPA treatment decreases RhoGDI-alpha gene expression, which points to apoptosis by JNK activation through a MAPKs-dependent pathway. A strong association between RhoGDI-alpha and Rac1 activation during MPA-induced apoptosis is also consistent with apoptosis through JNK. Suppression of RhoGDI-alpha using siRNA and gene over-expression both affected the cell death rate, consistent with Rac1 activation and downstream activation of MAPKs signaling. We confirmed that Rac1 protein mediates the interaction between RhoGDI-alpha and JNK signaling. We conclude that MPA-induced cell death in primary beta-cells and an insulin-secreting cell line proceeds through RhoGDI-alpha down-regulation linked to Rac1 activation, with subsequent activation of JNK. The RhoGDI-alpha/Rac1/JNK pathway may present a key to intervention in MPA-induced islet apoptosis.ope

Functional improvement of pig islet with exocrine encapsulation

Porcine-specific obstacles in islet isolation frequently result from the low purity or contamination with exocrine tissues. We implemented a new technique involving as capsulation of islets with excess exocrine tissue as a beneficial material to address those difficulties. Pig islets were hand-picked as high purity (HI) or low purity (LO) islets containing significant amounts of exocrine tissue. We performed static (ST) or shaking (SK) cultures of HI and LO islets. Islet function was examined after 24 hours by a glucose challenge test. Insulin secretion into the culture media was continuously measured using ELISA during a 6-day culture period. Islet function after 24 hours exhibited better maintenance under SK than ST culture as assessed by a stimulation index. The ideal islet morphology was seen in LO islets at 3 days of SK culture with typical islet shapes of a smooth surface and a spherical configuration. In contrast, typical islet morphology was not observed in HI islets under SK culture; maintenance of typical spherical appearances was difficult. Insulin secretion from LO islets under SK culture was higher than under other conditions during the 6-day period. Under SK culture conditions, exocrine-encapsulated LO islets showed enhanced islet function by condensing loose islet aggregates into firm spheroids with native exocrine tissues as a natural scaffold.ope

Cellular function of RhoGDI-α mediates the cycling of Rac1 and the regulation of pancreatic beta cell death

Mycophenolic acid (MPA) is an immunosuppressive agent that is widely used in clinical therapy, including pancreas and islet transplantation. Previously, we showed that MPA induces significant apoptosis of insulin-secreting cells by downregulating RhoGDI-α and increasing JNK expression. In this study, we investigated Rac1 directly associated with RhoGDI-α during MPA-induced apoptosis in INS-1E cells (an insulin-secreting cell line). Cells were treated with MPA for 24 and 36 hours. Immunoprecipitation was used to examine physical interactions between RhoGDI-α and Rac1. Activation and immunoprecipitation assays showed expressions of Rac1 and RhoGDI-α to be directly correlated. Rac1 binding to RhoGDI-α decreased after MPA treatment, and Rac1 was induced and subsequently activated by MPA. We concluded that this novel RhoGDI-α/Rac1/JNK pathway induced apoptosis of transplanted islet cells after MPA treatment.ope

Functional evaluation of chondrocyte sheeting immunodelusive immunoisolated bioartificial pancreas

In islet transplantation, encapsulation of immunoisolated islets may provide a way to protect the graft from immune attacks with no immunosuppression. To develop an immunodelusive immunoisolated bioartificial pancreas (BAP), chondrocyte sheets were prepared by cell sheet engineering. We made an immunoisolated BAP encapsulated with rodent-derived chondrocyte sheets and then evaluated its function. Sprague-Dawley rats were used as the source of auricular cartilage and chondrocytes were maintained and expanded by passages. Lewis rats were prepared for islet isolation. A 3-dimensional chondrocyte sheeting immunodelusive immunoisolated BAP (CSI-BAP) was created by multi-layering and unifying the chondrocyte sheets. Subsequently, islets were embedded between each multi-layer sheet. To evaluate the function of the CSI-BAP, a glucose challenge test was performed and secretion of insulin in the culture medium was measured by an enzyme-linked immunosorbent assay. When observed by phase-contrast microscopy, the CSI-BAP maintained close connections between chondrocyte sheets. Islets in the CSI-BAP maintained viability at day 10 and showed good insulin secretion, as revealed by a prompt reaction to increased concentrations of glucose at days 5 and 10. In long-term culture, the CSI-BAP maintained its ability to secrete insulin for 8 weeks. This BAP technology could be an important tool for successful islet transplantation without immunosuppressive drugs.ope