Insulin secretion is controlled by a complex set of factors. Although blood glucose levels serve as the major stimulus of insulin secretion in mammals, insulin release is also modulated by amino acids, catecholamines, glucagon, and other, intestinal hormones. The identification of factors that modulate insulin production has engendered much interest because of their potential importance in the altered dynamics of insulin secretion in response to glucose characteristic of maturity-onset diabetes mellitus. Decoding of the glucagon gene has uncovered two additional glucagon-like peptides encoded in proglucagon, the polypeptide precursor of glucagon. One ofthese peptides, glucagon-like peptide I, is processed from proglucagon in two forms, of 31 and 37 amino acids. We report that the smaller of the two glucagon-like peptides potently increases cAMP levels, insulin mRNA transcripts, and insulin release in cultured rat insulinoma cells. These results indicate that glucagon-like peptide I may be a physiologic modulator of insulin gene expression.

ABS1hACT Continuous cell lines that secrete both insulin and somatostatin were established by two cooprating groups of investigators from a serially transplantable, radiation-induced, rat islet cell tumor. The cell lines, named RIN-r and RIN-m, were initiated from tumors maintained in inbred rats or in athymic nude mice, respectively. The cultured cells are ppithelioid, free of fibroblast.contamination, and can be cloned.They have a hypodiploid chromosome number, are tumorigenic, and possess amine-handling properties, including high evels of L-dopa decarboxylase and formaldehyde-induced fluorescence. Preliminary analysis of clones revealed a spectrum of insulin secretion from undetectable to relatively high. These clonal cell lines provide important systems to study the biology of insulin and somatostatin. MATERIALS AND METHODS Origin of the cultures. Cultures were initiated from a transplantable islet cell tumor (6) induced by high-dose x-irradiation in an inbred NEDH (New England Deaconess Hospital) rat. The tumor was maintained by serial transplantation in NEDH rats. After nine transplants, it was successfully heterotransplanted into athymic nude mice, BALB/c background (ARS/Sprague-Dawley, Madison, WI). Continuous cell lines were derived either from rat transplants (Joslin group) or from nude mouse heterotransplants (NCI-VA group). These cell lines were named RIN-r and RIN-m, respectively.Establishment of RIN-r Cell Line. Tumors were removed aseptically from recipient rats and cut into small fragments The tumor cells were separated from the connective tissue stroma by washing the fragments with tissue culture medium 199 containing 0.1% fetal bovine serum, 16.5 mM glucose, and 400 units of penicillin per ml. The culture medium used throughout the remainder of the procedure was similar, but contained 10% fetal bovine serum. Erythrocytes present in the original cell suspension were removed by centrifugation (750 X g, 10 min) on a discontinuous gradient consisting of a solution of 25% di-

A transplantable insulinoma was developed in inbred albino rats of the NEDH strain. The original tumor, 1 cm in diameter, was removed from the pancreas of a male parabiont 566 days following 1000 rads (10 J/kg) of total body x-irradiation. The time required for implanted fragments to grow to 0.5-1.5 cm in diameter decreased from 5-8 months in the first generation to 2-5 months in the seventh generation. Successful transplantation in male animals followed for 4 or more months after transplantation was significantly greater than in female animals [ollowed for a similar period of time (96%to versus 69%). Lidt and electron microscopy revealed that the tumors consisted predominantly of well anulated beta cells. Ultrastructural studies also showed smal numbers of Dcells. Tumor extracts contained an average of 223 units of immunoreactive insulin and 25.9 fg of immunoreactive somatostatin per gram wet weight of tissue. Tumors generally produced increasingly profound hypoglycemia within 24 months following transplantation, with plasma glucose levels frequently falling to 40 mg/100 ml or lower prior to death. Removal of tumors from chronically hypoglycemic animals resulted in transient rebound hyperglycemia with plasma glucose levels above 300mg/100mlwithinthefirst24 rand a gradual decline to normal levels of 129 mg/100 ml in 24 days. These observations correlated with findings of marked atropy and degranulation of the beta cells in the pancreata of tumor-bearing animals, and with gradual return of normal light microscopic morphology following-tumor removal.Limitation of starting material has proven to be a serious obstacle in several important areas of pancreatic beta cell research. Although methods are available for isolating pancreatic islets by collagenase digestion and Ficoll gradient centrifugation, such procedures at best yield only a few milligrams of material (1, 2). This has made the preparation of subcellular fractions and the isolation of other constituents of the beta cell extremely difficult. Although a transplantable insulinoma developed several years ago in the hamster has provided relatively large amounts of tissue, the predominant cell types in this tumor remain unclear because the insulin content is extremely low and light microscopic sections fail to stain with appropriate aldehyde-type stains (3, 4).The present studies involve experiments with a recently developed radiation-induced transplantable rat insulinoma that has made available gram quantities of well-granulated beta cells with high insulin content (5). This material has already proven valuable for the isolation of messenger RNA for insulin synthesis (6, 7) and for the initiation of studies towards the development of a permanent line of insulin-producing cells (8, 9). The present report describes the method used to develop and to transplant this tumor, and its morphology, hormone content, and physiologic effects on host animals. The actions exerted by this insulinoma on the recipient's own pancreatic islet cells are also described.MATE...

Multiple small injections of streptozotocin produce a delayed, progressive increase in plasma-glucose in mice within 5-6 days after the injections, in association with pronounced insulitis and induction of type C viruses within beta cells. Multiple subdiabetogenic doses of streptozotocin in rats and multiple injections of another beta cell toxin, alloxan, in mice did not induce insulitis although hyperglycemia followed the injection of larger quantities of both agents. In mice, the prior injection of 3-O-methyl-D-glucose (3-OMG) or nicotinamide attenuated the diabetic syndrome produced by streptozotocin; however, 3-OMG was more protective. Rabbit antimouse lymphocyte serum, alone, provided partial protection but, when given together with either 3-OMG or nicotinamide, effectively prevented the streptozotocin-induced diabetic syndrome. Cessation of these preventive treatments was followed by the appearance of insulitis and diabetes. These findings suggest that multiple injections of streptozotocin induce, in susceptible hosts, the triad of direct beta cell cytotoxicity, virus induction within beta cells, and cell-mediated autoimmune reaction. These factors, acting separately or in concert, appear to induce a destructive insulitis and severe diabetes. The relative importance of each component and the factors governing host susceptibility remain to be clarified. Streptozotocin [SZ; 2-deoxy-2(3-methyl-3-nitrosoureido)-Dglucopyranose] is a broad-spectrum antibiotic with oncolytic, oncogenic, and diabetogenic properties (1-4). The diabetogenic action is mediated by selective destruction of pancreatic beta cells and has been widely utilized as a method for inducing diabetes mellitus in experimental animals and for treatment of malignant beta cell tumors and other neoplasms in humans. To produce diabetes, SZ is conventionally administered as a single injection. SZ is cleared from the bloodstream rapidly (serum half-life, 15 min) (5); beta cell necrosis can be detected by electron microscopy within hours after SZ injection (6-8). Elevated blood glucose levels are demonstrable within 1-2 days, and dissolution and phagocytosis of necrotic cells are observed histologically after 3 days (3, 6). In rats and mice, the administration of a single subdiabetogenic dose produces only mild histologic alterations without evidence of significant hyperglycemia when compared with buffer-injected control animals (9, 10).We have recently reported (10) that the administration of multiple (five) subdiabetogenic doses of SZ to Charles River Laboratory (CD-1) mice, either intravenously or intraperitoneally, produced a delayed but progressive increase in blood glucose with levels of 350450 mg/100 ml within 5-6 days after the last injection. Morphologically, the pancreatic islets revealed pronounced insulitis with infiltrating lymphocytes and macrophages, architectural distortion, and beta cell necrosis. There was a subsequent decrease in inflammation within the remaining islets which were small and composed almost exclusively of non-bet...

ABSTRACr We have cloned double-stranded cDNA copies of a rat preproinsulin messenger RNA in Escherichia coli X1776, using the unique Pst endonuclease site of plasmid pBR322 that lies in the region encoding amino acids 181-182 of penicillinase. This site was reconstructed by inserting the cDNA with an oligo(dG)oligo(dC) joining procedure. One of the clones expresses a fused protein bearing both insulin and penicillinase antigenic determinants. The DNA sequence of this plasmid shows that the insulin region is read in phase; a stretch of six glycine residues connects the alanine at position 182 of penicil inase to the fourth amino acid, glutamine, of rat proinsulin. Can the structural information for the production of a higher cell protein be inserted into a plasmid in such a way as to be expressed in a transformed bacterium? To attack this problem, we used as a model rat insulin, an interesting protein that can be identified by immunological and biological means.Although mature insulin contains two chains, A and B, it is the product of a single longer polypeptide chain. The hormone is initially synthesized as a preproinsulin structure (1, 2). A hydrophobic leader sequence of 23 amino acids at the amino terminus of the nascent chain is cleaved off, presumably as the polypeptide chain moves through the endoplasmic reticulum (2-4), producing a proinsulin molecule. The proinsulin chain folds up and then the C peptide is cleaved from its middle (5). Thus each of the two (nonallelic) insulin genes in the rat (6-8) encodes a polypeptide 109 amino acids long, whose initial structure is NH2-leader sequence-B chain-C peptide-A chain.Ullrich et al. (9) have cloned double-stranded cDNA copies of rat preproinsulin mRNA isolated from pancreatic islets and determined sequences covering much of those two genes. We have made double-stranded cDNA copies of mRNA from a rat insulinoma (10) and cloned these in the Pst (Providencia stuartli endonuclease) site of pBR322 (11), which lies within the penicillinase gene.The Escherichia coil penicillinase is a periplasmic protein, the gene for which was recently sequenced (12). Penicillinase is synthesized as a preprotein with a 23 amino acid leader sequence (12, 13), which presumably serves as a signal to direct the secretion of the protein to the periplasmic space, and is removed as the protein traverses the membrane. Insertion of the structural information for insulin into the penicillinase gene should cause expression of the insulin sequence as a fusion product transported outside the cell.MATERIALS AND METHODS Bacterial Strains. E. coli K-12, strain HB101 [hsm-, hrs-, recA-, gal-, pro-, strr (14)] was initially obtained from H.Boyer. E. coli K-12 strain X1776 (15) (F-, tonA53, dapD8, RNA Purification. An x-ray-induced, transplantable rat beta cell tumor (10) was used as source of preproinsulin mRNA. Tumor slices (20 g per preparation) were homogenized, and a cytoplasmic RNA (about 2 mg/g of tissue) was purified from a postnuclear supernatant by Mg2+ precipitation (17), followed by extra...