Establishment of glucagon-producing cells by cell hybridization.

Glucagon-producing cell lines were established by fusing pancreatic islet cells of adult hamster and 6-thioguanine-resistant hamster insulinoma cells. Under phase-contrast microscopy, the morphology of cultured hybrid cells was intermediate between those of the parental cells. The hybrid cells contained A-like granules, though few in number, and were stained with anti-glucagon antibody. The mode of chromosome number decreased to 78 or 79 by 3 mo after hybridization in comparison with the expected chromosome number of the heterokaryon of 104, and showed a minute decrease in 4 of 6 cell lines after 6 mo. The population doubling time ranged from 24 to 38 h, while that of parental insulinoma cells was 22.8 h. There was no correlation between the expression of cellular function and the stability of chromosome number or the length of population doubling time. The capacity of glucagon secretion was between that of the parental cells. The glucagon secreted into the medium, as assayed by the glucagon-specific antibody, was 0.6-2.5 ng/10(6) cells for 2 h, which was about 40% of total glucagon-like immunoreactivity secreted. Secretion of glucagon was not affected by high concentration of glucose, was markedly increased by theophylline, and was suppressed by exogenous insulin. All of the hybrid cells produced tumors on transplantation 6 mo after hybridization. The tumor-bearing hamsters exhibited high levels of plasma glucagon and blood glucose as well as a high level of serum insulin.

Effect of phorbol esters on glucagon secretion from a glucagon-secreting clonal cell line. Synergistic effects of A23187 and theophylline.

The cell line In-R1-G9 is one of the clones from the hamster insulinoma cell line, In-111-R1, and it produces glucagon. Phorbol esters markedly enhanced glucagon secretion and the stimulatory effect was found to be correlated to their biological activity as tumor promoters. At a concentration of 200 nM, 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated glucagon secretion 13-fold more than the control in 10 min. The effect of TPA was not influenced by actinomycin D, cycloheximide, colchicine or vincristine. Depletion of calcium from the incubation medium inhibited TPA-induced glucagon secretion by approximately 50% and dibucaine also suppressed glucagon secretion to 67.4%. An addition of A23187 to TPA induced 150% enhancement over the TPA-stimulated glucagon level, and the maximum secretory response was observed when the cells were stimulated with the simultaneous addition of TPA, A23187 and theophylline.

Isolation of glucagon-secreting cell lines by cloning insulinoma cells.

Six glucagon-secreting cell lines designated as In-R1-G1, -G3, -G7, -G9, -G10, and -G11 were isolated from insulinoma cells (In-111-R1) by single cell cloning. A small amount of insulin was also detectable in the incubation medium when hormone secretion was stimulated by the addition of arginine or theophylline. These cell lines grew as monolayers and the population doubling times varied from 16.8 to 28.8 h. Karyologically these clones were aneuploid and the modes of chromosome numbers were 61 to 70. Electron microscopic examination of one of these clones showed that these cells contained moderately developed Golgi apparatus and a few secretory granules, which more or less resembled alpha-cell granules. By gel filtration study of the incubation medium, glucagon and glucagonlike material were eluted. The molecular weight of the latter was approximately 9000, which suggested the concomitant secretion of proglucagon into the medium. The levels of secreted glucagon in basal state were 0.3 to 3.0 ng/10(6) cells/2 h. Glucagon secretion was markedly enhanced in the presence of amino acids. Glucagon secretion increased slightly in the presence of high concentration of glucose in Hanks' balanced salt solution; however it was not affected by the varying concentrations of glucose when the cells were incubated in complete media with amino acids. Glucagon secretion was also stimulated by the addition of theophylline. These clonal cell lines seem to provide a useful tool for investigating the mechanism of glucagon secretion.