SAT-160 Fetuin-a Secreted From Pancreatic β-cells Plays Significant Role In Macrophage Accumulation And Inflammation In The Islets During Hyperlipidemic Condition

Abstract

Inflammation in the pancreatic islets by means of increased macrophage migration and elevated levels of pro-inflammatory cytokines in the circulating serum occurs during Type 2 Diabetes. Another mechanism underlying increase in inflammation is polarization of macrophages from anti-inflammatory M2 to pro-inflammatory M1 form. Whether such changes occur in the infiltrating macrophages in the islets and what were the factors leading to these changes arte yet unknown. Fetuin-A, a glycoprotein known to be secreted from liver and adipocytes has also been found to be secreted from pancreatic β-cells by our group recently. Role of fetuin-A in macrophage polarisation has been worked upon but still more work is yet to be done. Our focus of interest was whether the β-cell derived fetuin-A has any role in polarisation of macrophages migrating to the islets leading to increase in inflammation. For in vivo model we used high fat diet mice model and for in vitro studies we used MIN6 pancreatic β cell line and RAW 264.7 murine macrophage cell line. In the in vivo mice model immunohistochemistry studies revealed increase in number of M1 macrophages in the pancreatic islets and fetuin-A secretion in comparison to the control standard diet fed mice (CD11c, arginase, F4/80). Also there was decrease in islet mass, specifically in number of β-cells. Fetuin-A KD mice using vivo morpholino showed decrease in infiltrating M1 macrophages in the islets as well improved β-cell survival. For replicating hyperlipidemic condion in vitro we treated the MIN6 cells with various doses of palmitate (0, 0.25, 0.50 and 0.75 mM) and assessed the increase in number of infiltrating RAW 264.7 macrophages (4 folds) in a boyden chamber system along with increasing level of fetuin-A (3 folds) in the media as determined by ELISA. Next the effect of this β-cell derived fetuin-A was observed upon macrophage polarisation. Originally RAW264.7 is anti-inflammatory M2 type. The cells were treated with fetuin-A secreted from β-cells, palmitate alone and LPS plus TNFα (positive control), while a set of macrophages were left untreated as negative control. The cells were collected and subjected to qualitative PCR for identifying the expression of M1 and M2 markers like CD11c, arginase, F4/80 and it was found that the pro-inflammatory markers increased in fetuin-A treated RAW 264.7 cells in similar levels like that of LPS+TNFα levels. These results indicated possible role of β-cell derived fetuin-A in macrophage polarisation in the islets. Another important point was that β-cells also express MCP1 another chemoattractant. Blocking of MCP1 by si-RNA decreased macrophage migration a little but expression of fetuin-A was enough for significant number of macrophages to migrate, however blocking fetuin-A by si-RNA in MIN6 cells decreased the number of migrating macrophages significantly.