2 DISTINCT SIGNALING PATHWAYS ACTIVATED BY ACTIVIN-A IN GLUCOSE-RESPONSIVE PANCREATIC BETA-CELLS LINES

Activin A stimulates insulin secretion in pancreatic beta-cells by a c alcium-dependent mechanism. The present study was conducted to further characterize the effects of activin A in two glucose-responsive insul inoma cell lines, MIN6 and HIT-T15 cells. In HIT-T15 cells, activin A evoked an increase in cytoplasmic free calcium concentration, stimulat ed insulin secretion, maintained glucose responsiveness of the cells a nd inhibited DNA synthesis. However, activin A did not have any effect in MIN6 cells. Measurement of I-125-labeled activin A binding in MIN6 cells revealed that the number of binding sites was markedly reduced, suggesting that the refractoriness was due, at least partly, to the r educed numbers of the activin receptor. Stable transfectants of MIN6 c ells that overexpressed the type II activin receptor were then develop ed. The transfected cells (MIN6-ActR cells) expressed ten times more I -125-labeled activin A-binding sites than parental cells and the appar ent K-d was 1.15 nM, which was nearly identical to that in parental ce lls. Affinity cross-linking in MIN6-ActR cells showed that a 90 kDa ty pe II receptor as well as a 52 kDa protein, presumably follistatin, wa s markedly labeled with I-125-labeled activin A. Although MIN6-ActR ce lls expressed significant numbers of activin receptors, activin A did not induce immediate calcium-dependent responses in these cells. In co ntrast, activin A was capable of inducing long-term effects in MIN6-Ac tR cells; thus, reduction of the glucose concentration in culture medi um from 25 to 5.5 mM for 4 days resulted in a remarkable loss of insul in response to glucose stimulation but his decline in response to gluc ose was prevented by the addition of activin A during culture. In addi tion, activin A inhibited DNA synthesis in MIN6-ActR cells. Hence, alt hough activin A did not induce calcium-dependent responses, it evoked some calcium-independent effects in MIN6-ActR cells. Taken together, a ctivin A elicits various effects in beta-cells by both calcium-depende nt and -independent mechanism.