ERp46 is reduced by high glucose and regulates insulin content in pancreatic beta-cells.

Our studies focus on ERp46, an endoplasmic reticulum (ER) component, and analyze its involvement in glucose toxicity and in insulin production. Differences in pancreatic beta-TC-6 cell proteome under conditions of low vs. high glucose were examined by proteomic approaches, including two-dimensional gel electrophoresis, image analysis, and mass spectrometry. Among differentially expressed proteins, ERp46, a novel endoplasmic reticulum component, was examined further. The expression of ERp46 in pancreatic sections was analyzed by immunocytochemistry, and high glucose-induced alterations of expression were evaluated in cultured beta-cells, in isolated pancreatic islets, and in the pancreas of db/db diabetic animals. Inhibition of ERp46 expression by siRNA was performed to study its role in insulin production, in secretion, and in ER stress. Proteomic analysis led to identification of 46 differentially expressed spots corresponding to 23 proteins. Since ERp46 is a novel protein with a possible crucial role in secretory cells, we further analyzed its role in beta-cell function. ERp46 expression is reduced in high glucose concentration in beta-TC-6 cells and in isolated murine islets. Further analysis revealed high expression of ERp46 in pancreatic islets compared with exocrine tissue. Interestingly, a marked decrease in ERp46 expression was found in the pancreatic islets of db/db mice. Most importantly, siRNA-mediated knockdown of ERp46 in cultured beta-cells led to a significant decrease in the insulin content; however, no alterations in insulin mRNA levels were observed under these conditions. In addition, reduced expression of ERp46 by siRNA increased the expression of CHOP and peIF2a, indicating development of ER stress. We conclude that ERp46 may be an important component in the phenomenon of "glucose toxicity" involved in insulin production at the posttranslational level.

Altered expression of calreticulin during the development of fibrosis.

Tissue damage following injury leads to inflammation and fibrosis. To understand the molecular mechanisms and the proteins involved in the fibrotic process, we used the well-established unilateral ureteric obstruction rat model and we analyzed the alterations at early and late time intervals using a classical proteomic approach. Data analysis demonstrates a correlation between calreticulin up-regulation and progression of fibrosis. Calreticulin is involved in Ca++ homeostasis but has not been previously implicated in animal models of fibrosis. Proteomic analysis consistently revealed up-regulation of calreticulin in both early and late time intervals. These findings were further confirmed by biochemical and morphological approaches. Next, animal models of lung fibrosis (bleomycin-induced) and heart fibrosis (desmin-null) were examined. In the lung model, calreticulin expression was up-regulated from early time intervals, whereas in the heart model no change in the expression of calreticulin was observed. In addition, TGF-beta, a well known major contributing factor in several fibrotic processes, was found to up-regulate calreticulin in cultured human proximal tubule epithelial cells. The above observations suggest that calreticulin might be involved in fibrotic processes; however the mechanism(s) underlying its possible involvement are yet unresolved.