Localization of insulin in subcellular fractions of saurian endocrine pancreas

Abstract

The secretory granules of the three major cell types in the splenic pancreas of the lizard Anolis carolinensis differ in their size distribution and electron density. Such differences might be useful for isolating a more homogeneous population of beta secretory granules than has been achieved with islet cells from other vertebrates. The localization of insulin during differential centrifugation and discontinuous sucrose density gradient centrifugation was determined by radioimmunoassay of the fractions. The behavior of beta secretory granules of Anolis during differential centrifugation suggests differences in physical characteristics between anolian and rat beta granules. Similarly, the anolian beta granules exhibited a greater apparent density during centrifugation in sucrose gradients. Differential centrifugation resulted in a fraction containing the majority of insulin (about 40%), but there was significant contamination with mitochondrial, microsomal, and ribosomal components. Discontinuous sucrose density gradient centrifugation resulted in a considerable reduction of the apparent mitochondrial contamination, but the occurrence of microsomal and ribosomal elements was still widespread. After density gradient centrifugations, the ultrastructural appearance of the beta granules in the insulin-rich fractions was variable. With the sucrose gradient employed many beta granules had more or less intact limiting membranes and the granules were generally similar to those observed in the whole pancreas. However, cores of granules, with and without surrounding matrix material, were frequently observed. The present findings suggest that future attempts to obtain isolated, intact beta granules from Anolis should include the possible utilization of size differences in the secretory granules of the islet cells in this species. In addition, an intact limiting membrane as a component of the beta granules should be a prerequisite for studies directed at assessing beta granule stability.