Postabsorptive hyperglucagonemia in patients with type 2 diabetes mellitus analyzed with a novel enzyme-linked immunosorbent assay.

AIMS/INTRODUCTION: The aims of the present study were to investigate the performance of a novel sandwich enzyme-linked immunosorbent assay (ELISA) for measuring glucagon (1-29) with monoclonal antibodies against both the C- and N-terminal regions of glucagon (1-29), and to analyze the differences in plasma levels and responses of glucagon (1-29) to oral glucose loading in normal glucose tolerance (NGT) subjects and patients with type 2 diabetes mellitus. MATERIALS AND METHODS: The cross-reactivity against proglucagon fragments using the ELISA kit and two types of conventional radioimmunoassay (RIA) kits was evaluated. A 75-g oral glucose tolerance test was carried out with NGT subjects and patients with type 2 diabetes mellitus, and the glucagon (1-29) concentration was measured using three types of kit. RESULTS: The ELISA kit clearly had the lowest cross-reactivity against miniglucagon (19-29) and glicentin (1-61). The oral glucose tolerance test was carried out with 30 NGT and 17 patients with type 2 diabetes mellitus. The glucagon (1-29) levels measured by the ELISA kit after glucose loading were significantly higher at all time-points in the type 2 diabetes mellitus group than in the NGT group. However, the glucagon (1-29) levels measured by one RIA kit were significantly higher in the NGT group, and those measured with the other RIA kit were approximately the same among the groups. CONCLUSIONS: The novel sandwich ELISA accurately determines plasma glucagon (1-29) concentrations with much less cross-reactivity against other proglucagon fragments than conventional RIA kits.

Glucagon-reactive islet-infiltrating CD8 T cells in NOD mice.

Type 1 diabetes is characterized by T-cell-mediated destruction of the insulin-producing ß cells in pancreatic islets. A number of islet antigens recognized by CD8 T cells that contribute to disease pathogenesis in non-obese diabetic (NOD) mice have been identified; however, the antigenic specificities of the majority of the islet-infiltrating cells have yet to be determined. The primary goal of the current study was to identify candidate antigens based on the level and specificity of expression of their genes in mouse islets and in the mouse ß cell line MIN6. Peptides derived from the candidates were selected based on their predicted ability to bind H-2K(d) and were examined for recognition by islet-infiltrating T cells from NOD mice. Several proteins, including those encoded by Abcc8, Atp2a2, Pcsk2, Peg3 and Scg2, were validated as antigens in this way. Interestingly, islet-infiltrating T cells were also found to recognize peptides derived from proglucagon, whose expression in pancreatic islets is associated with α cells, which are not usually implicated in type 1 diabetes pathogenesis. However, type 1 diabetes patients have been reported to have serum autoantibodies to glucagon, and NOD mouse studies have shown a decrease in α cell mass during disease pathogenesis. Our finding of islet-infiltrating glucagon-specific T cells is consistent with these reports and suggests the possibility of α cell involvement in development and progression of disease.