Characterization of MANF expression and secretion under serum and glucose deprivation in vitro

Abstract

Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) was discovered to promote neuronal survival in rodent models of Parkinson’s disease (PD) and cerebral ischemia. MANF belongs to a novel evolutionary conserved family of neurotrophic factors (NTFs) and it is an endoplasmic reticulum (ER) stress response protein. MANF was more recently shown to promote beta cell proliferation and survival and its levels in serum are increased at the onset of type 1 diabetes in children. MANF protein and mRNA expression is widespread in most human and mouse organs. However, MANF expression and secretion levels from cells depending on the availability of energy sources have not been previously studied and, study it was the main objective of the project. Characterization of MANF expression and secretion was done in vitro from mouse insulinoma 6 (MIN6) and human endothelial (EA.hy926) cell lines cultured under serum or glucose deprivation. Enzyme-linked immunosorbent assay (ELISA) specific for human and mouse MANF were used to characterize intracellular and secreted MANF protein levels. Moreover, MANF protein and ER stress markers were analyzed by using western blotting. The mRNA expression of MANF and ER stress markers was also analyzed using quantitative PCR method. The results indicated that intracellular MANF levels are up-regulated under glucose deprivation in MIN6 and EA.hy926 cell lines. However, secreted MANF levels increase when glucose concentration in the medium decreases only in beta pancreatic insulin producers cells (MIN6), in contrast to EA.hy926 cells, indicating that responses to glucose deprivation are different in the two cell lines. Furthermore, intracellular and secreted MANF levels increase when serum concentration is decreased in both MIN6 and EA.hy926 cell lines. The studied human endothelial cell line present increased levels of Grp78, an ER stress marker, after serum and glucose deprivation.