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Normal adult rat islets usually remained intact and encapsulated, even after many days in culture. In contrast, islets cultured in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1 mM) attached more readily to the surface of plastic culture dishes and almost uniformly formed monolayers of endocrine cells. The mechanism of this effect is not known but presumably involves increases in cellular cyclic AMP content. Fibroblast growth did not appear to be stimulated by the inhibitor. These adult pancreatic endocrine monolayer cultures can be produced readily and provide useful preparations for further morphological and biochemical studies of factors affecting the differentiation, growth, and regenerative capacity of islet cells.

SUMMARY Recent studies have established that limited intracellular proteolysis may participate at several stages in the biosynthesis of many small polypeptide hormones, as exemplified by insulin. Conversion of proinsulin to insulin by trypsin-and carboxypeptidase B-like enzymes occurs in the pancreatic B cell after the peptide has been sequestered from the cytosolic compartment and transported to the Golgi area. An earlier role for proteolysis concerns the conversion of the initial translation product, preproinsulin, to proinsulin during its compartmentalization in the rough endoplasmic reticulum. Preproinsulin, a methionine-initiated peptide which contains a hydrophobic 24-residue NH 2 -terminal extension, was originally identified as the product of the cell-free translation of insulin mRNA, but more recently it has also been detected in small amounts in intact rat islets. Its rapid formation and disappearance during pulse-chase studies are consistent with its role as a biosynthetic precursor and with the proposal that the prepeptide region serves as a leader sequence in the formation of the ribosome-membrane junction and in the vectorial discharge of the peptide. A model for segregation is presented, based on important structural features and physical properties of the prepeptides which may enable these to enter and span the microsomal membrane. Studies on insulin mRNA obtained from transplantable islet cell tumors indicate that the major component contains approximately 600 nucleotides and possesses a 5′ “cap” structure as well as a 3′ polyadenylate “tail”. A closer examination of the insulin tumor mRNA has also revealed two larger polyadenylated RNA fractions having apparent molecular weights of 280,000 and 360,000. These forms, which hybridize to the rat insulin genes and which were shown by ribonuclease T 1 digests to share nucleotide sequences with the major mRNA component, may represent precursors of the mature template. These results indicate that several sequential stages of macromolecular processing are required in the formation of the secreted hormone.

Newly synthesized rat islet proteins have been analyzed by polyacrylamide slab gel electrophoresis and fluorography. A minor component having an apparent molecular weight of 11,100 was identified as preproinsulin by the sensitivity of its synthesis to glucose, the pattern of NH2-terminal leucine residues, and the rapidity of its appearance and disappearance during incubation of islets or islet cell tumors. A small amount of labeled peptide material which may represent the excised NH2-terminal extension of preproinsulin or its fragment was also detected. The kinetics of formation and processing of the preproinsulin fraction were complex, consisting of a rapidly turning over component having a half-life of about 1 min and a slower minor fraction that may have bypassed the normal cleavage process. The electrophoretic resolution of the preproinsulin and proinsulin fractions into two bands each is consistent with the presence of two closely related gene products in rat islets rather than intermediate stages in the processing of these peptides.