Beta-cell gene expression and functional characterisation of the human insulinoma cell line CM.

Animal insulinoma cell lines are widely used to study physiological and pathophysiological mechanisms involved in glucose metabolism and to establish in vitro models for studies on beta-cells. In contrast, human insulinoma cell lines are rarely used because of difficulties in obtaining and culturing them for long periods. The aim of our study was to investigate, under different experimental conditions, the capacity of the human insulinoma cell line CM to retain beta-cell function, particularly the expression of constitutive beta-cell genes (insulin, the glucose transporters GLUT1 and GLUT2, glucokinase), intracellular and secreted insulin, beta-cell granules, and cAMP content. Results showed that CM cells from an early-passage express specific beta-cell genes in response to glucose stimulation, in particular the insulin and GLUT genes. Such capacity is lost at later passages when cells are cultured at standard glucose concentrations. However, if cultured at lower glucose concentration (0.8 mM) for a longer time, CM cells re-acquire the capacity to respond to glucose stimulation, as shown by the increased expression of beta-cell genes (insulin, GLUT2, glucokinase). Nonetheless, insulin secretion could not be restored under such experimental conditions despite the presence of intracellular insulin, although cAMP response to a potent activator of adenylate cyclase, forskolin, was present indicating a viable system. In conclusion, these data show that the human insulinoma cell line CM, at both early-passage and late-passage, posseses a functional glucose-signalling pathway and insulin mRNA expression similar to normal beta-cells, representing, therefore, a good model for studies concerning the signalling and expression of beta-cells. Furthermore, we have previously shown that it is also a good model for immunological studies. In this respect it is important to note that the CM cell line is one of the very few existing human beta-cell lines in long-term culture.

Immune reactivity of diabetes-associated human monoclonal autoantibodies defines multiple epitopes and detects two domain boundaries in glutamate decarboxylase.

Autoreactive islet cell Abs (ICA) accompany the pathogenic destruction of pancreatic beta cells in insulin-dependent diabetes mellitus (IDDM). Human monoclonal ICA (MICA 1-6), previously derived from a DR1/DR7-positive newly diagnosed diabetic patient, recognized the islet cell autoantigen glutamate decarboxylase 65 (GAD65) and defined two distinct conformational (MICA 1/3 and MICA 4/6) and one linear (MICA 2) autoimmune epitopes in this molecule. We have isolated 4 new ICA-reactive B cell lines, one from a DR4/DR11-positive newly diagnosed IDDM patient (MICA 7) and three from a DR3 homozygous patient with both IDDM and Graves' disease (MICA 8-10). Like MICA 1-6, MICA 7-10 are specific for GAD65, suggesting that GAD65-reactive B cells dominate the ICA response in IDDM. Comparative analysis of MICA 1-6 and MICA 7-10, using GAD65 mutants and blocking experiments, showed that MICA 7-10 define three novel conformational autoimmune epitopes in GAD65. Further structural analysis of the MICA 1-10 epitopes revealed two distinct and one overlapping region of epitope clusters. Thus, the C-terminal region, defined by amino acids 450 to 570, harbors the conformational MICA1/3 and MICA 7 epitopes as well as the linear epitope of MICA 2 (amino acids 506-531). The MICA 4/6 and MICA 10 epitopes are located in the middle region of the molecule defined by amino acids 245 to 449, whereas the N-terminal region contributes only to the MICA 8/9 epitopes (encompassed in amino acids 39-585). MICA 1-6, 7, and 8-10, derived from three IDDM patients of different HLA haplotypes, define six different epitopes in GAD65 and represent tools to determine the spectrum, possible HLA association, and temporal order of epitope recognition in IDDM.