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Endogenous glucagon-cells are necessary for the glucose-responsiveness of the HIT-T15 hamster β-cell line

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Title: Endogenous glucagon-cells are necessary for the glucose-responsiveness of the HIT-T15 hamster β-cell line
Author: Väänänen, Jeffrey Eric
Degree Master of Science - MSc
Program Physiology
Copyright Date: 1993
Abstract: The role of glucagon in glucose-stimulated insulin release was investigated using the hamster insulinoma cell line, HIT-T15, as a model of the pancreatic β-cell. Glucose-stimulated insulin release from HIT-T15 cells was concentration-dependent over a range of glucose concentrations between 0 and 15 mM, with immunoreactive-insulin release (IRI) rising significantly above the basal level (zero glucose) when cells were exposed to glucose concentrations of 5, 10 and 15 mM (p < 0.02). Minimum (zero glucose) and maximum (15 mM glucose) insulin release was 1.6 ± 0.5 % and 12.0 ± 2.9 % of total cell content (TCC), respectively. In HIT-T15 cells, glucagon secretion was 5-9 % of total cell content during a 1 h release experiment. There was no significant difference between glucagon released in the presence of zero- or high (15 mM)-glucose. The addition of a glucagon antibody completely abolished glucose-stimulated insulin release, while antibodies raised against somatostatin and glucose-dependent insulinotropic polypeptide (GIP) had no effect. When HIT-T15 cells were incubated in conditions of zero-glucose and glucose plus glucagon-Ab there was no significant difference in insulin release (p >0.05). Furthermore, glucagon-Ab inhibited glucose-stimulated insulin secretion over the full range of glucose responsiveness (5 to 15 mM; p < 0.03). The abolition of glucose-stimulated insulin secretion by an antibody to glucagon indicates that glucose-stimulated insulin secretion is dependent on glucagon, probably acting via a receptor-dependent pathway. HIT-T15 cells that were co-cultured with glucagon producing InR1-G9 cells, displayed basal insulin release that was elevated, with an absence of glucose potentiation. HIT-T15 cells were immunoperoxidase stained for the following peptide hormones: insulin, somatostatin, glucagon, glucagon-like polypeptide-I, GIP, secretin, pancreatic polypeptide (PP) and peptide-tyrosine-tyrosine (PYY). Staining was positive for all peptide hormones tested except somatostatin, secretin and GIP. Insulin, glucagon and PYY were the dominant peptides. Double staining of HIT-T15 cells for insulin and glucagon demonstrated colocalization of both peptides within single cells. Additionally, HIT-T15 cells were immunoreactive for the cell surface adhesion molecule uvomorulin. Immunocytochemical studies of InR1-G9 cells demonstrated the presence of glucagon, GLP-I and PP, with PP immunoreactivity present in every cell. However, no immunoreactivity for insulin, somatostatin, GIP, secretin, PYY or uvomorulin was seen. Equal amounts of HIT-T15 and InR1-G9 cells were co-cultured, and stained with fluorescent and immunoperoxidase techniques. HIT-T15 cell clusters appeared to be surrounded by InR1-G9 cells. These cell lines appeared healthy when cultured together, in spite of the fact that these two cell lines are derived from different strains of hamsters, by different vectors. Electron microscopic examination revealed differences in HIT-T15 cell insulin secretory granule number with different culture conditions, including: low-glucose media (0.8 mM), high-glucose media (11.1 mM) and high-glucose media (9.1 mM) plus InR1-G9 cells. Most importantly, HIT-T15 cells cultured in high-glucose (11.1 mM) were extensively granulated, while those cultured in high-glucose (9.1 mM) in the presence of InR1-G9 cells were agranular. In conclusion, glucose-stimulated insulin secretion from the HIT-T15 cell line is glucagon-dependent, and endogenous glucagon containing cells (HIT-T15-G) can provide the glucagon. The addition of glucagon secreting cells (in large numbers) led to chronic stimulation of HIT-T15 cell secretion, and secretion could not be potentiated by glucose. Furthermore, co-culturing HIT-T15 cells with equal numbers of InR1-G9 cells led to a complete degranulation ofHIT-T15 cells, most likely by continuous stimulation. The HIT-T15 cells are a heterogeneous cell population, expressing multiple peptides, some of which are colocalized, as in the case of insulin and glucagon. InR1-G9 cells appeared to be relatively homologous with respect to peptide production. These cells extended neuron-like processes, which contained the majority of the secretory granules.
URI: http://hdl.handle.net/2429/1513
Series/Report no. UBC Retrospective Theses Digitization Project [http://www.library.ubc.ca/archives/retro_theses/]

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