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Renin and angiotensinogen have been previously found in the rat pancreas, and angiotensin receptors have been located in the apical domain of duct cells. To evaluate the possibility that angiotensin II could be generated within the duct system, we decided to determine whether angiotensinogen is present in rat pancreatic juice and the angiotensinogen-immunoreactive pancreatic cell types that could be responsible for its production. Angiotensinogen was detected in significant amounts by Western blotting in pancreatic juice collected from several individual rats. Different isoforms between plasma and pancreatic juice angiotensinogens were demonstrated by isoelectric focusing. Immunocytochemical experiments revealed angiotensinogen-immunoreactive cells at the periphery of the islets of Langerhans, and confocal microscopy demonstrated that most angiotensinogen-immunoreactive cells were glucagon-secreting cells. Secretion of angiotensinogen did not follow the regulated secretory pathway since it was absent from the glucagon-containing granules. This was confirmed by electron microscopy immunocytochemistry. Duct and acinar cells did not express angiotensinogen at an immunocytochemical detectable level. The present findings indicated an exocrine secretion of angiotensinogen by glucagon-secreting cells and suggest that one of the final targets of the local pancreatic renin-angiotensin system may be the duct epithelium.

Clusterin is a heterodimeric glycoprotein which has been shown to play important roles in programmed cell death and/or in tissue reorganization not only during embryonic development but also in damaged tissues. Recently, we reported the transient induction of clusterin in pancreatic endocrine cells during early developmental stages of islet formation. In the present study, we have investigated the expression of clusterin in pancreatic tissue of streptozotocin-treated rats which were undergoing extensive islet tissue reorganization due to degeneration of insulin beta cells. Clusterin was found in endocrine cells identified as glucagon-secreting alpha cells at the periphery of the islet. Using immunoelectron microscopy, clusterin-positive cells showed the typical ultrastructural features of pancreatic alpha cells. In addition, colocalization of clusterin and glucagon in the same secretory granules was shown by double immunogold labeling. These results imply that clusterin is a secretory molecule having endocrine and/or paracrine actions in parallel with glucagon. Further, we noted that clusterin expression was increased in pancreatic alpha cells during the process of beta cell death upon streptozotocin injection. The increase was significant as early as 1-3 h after streptozotocin treatment prior to any morphological alteration of islet beta cell and any manifestation of hyperglycemia. The expression of clusterin was steady-stately up-regulated during the process of islet reorganization caused by streptozotocin-induced cytotoxic injury. Therefore, we suggest that clusterin might be considered as a molecule induced by both embryonic development and drug-induced reorganization of the endocrine pancreas. Since clusterin expression is up-regulated in alpha cells, but not in beta cells undergoing degeneration, it may play a protective role against the cytotoxic insult.

Clusterin has been known to play important roles not only in remodeling damaged tissues, but also in tissue reorganization during embryonic development. In the present study, we have investigated the expression of clusterin in the endocrine pancreas during embryonic development. Although a weak immunoreaction was detected in some pancreatic primordial cells at day 14 of gestation, distinct clusterin expression was identified by immunocytochemistry and Northern blot analysis at the 16th day of gestation. Clusterin-producing cells, which corresponded to insulin-containing cells, accounted for the major portion of the developing islet of Langerhans up to 18 days of gestation. Thereafter, clusterin-producing cells display similar distribution and morphological features to glucagon-producing cells. Clusterin expressed in the pancreas was shown by Western blot analysis to be a disulfide-linked heterodimer of 70 kDa with an alpha-subunit of 32 kDa. During early developmental stages, however, we found that proteolytic internal cleavage of the clusterin molecule occurred from the 18th day of gestation. Only one 70 kDa band on the 16th day and two bands (32 kDa and 70 kDa) on the 18th day of gestation were detected by Western blot analysis even in reducing conditions, while only a single 32 kDa band was detected on the second day after birth. The levels of clusterin mRNA in the pancreas transiently increased from the 16th day of gestation to the second day after birth, during the period when active cellular reorganization takes place to form the classic cellular features of the islet. Among various tissue (kidney, brain, liver, heart, lung and pancreas) the levels of clusterin mRNA were the highest in the pancreas from the 18th day of gestation to the second day after birth. In contrast, the lowest expression was observed in adult pancreatic tissue. The higher expression of clusterin in developing pancreas must indicate its involvement in tissue organization during development.