The Potential Effects Of Autophagy Inhibition And Activation On The Differentiation In MIN6 Cells

Background:The pathogenesis of diabetes involoves two important steps including not sufficient islulin secretion of pancreas islet β cells and insulin resistance. Accelerating the proloferation of islet β cells and intruducing the regeneration of β cells as well as inhibiing the apoptosis ofβ cells are recent research topics. Recent studies have found that utophagy takes pivotal role on regulating β differenciation, inhibiting β cell apoptosis and homeostasis.Endocrine precursor ε cells and β cells derive from common endocrine precursor cellswhich keep the banlance of defferenciation via Inner mechanism. GhrelinIs expressed in theεcell,insulin is expressed in the β cell. Preliminery researches have found the imbalance of β cell differenciation in the ilets of newborn rates with inter uterine malnutrition. Therefore, we propose that understanding the mechanism of ε/βdifferentiationImbalance will bebefict for adjusting βcells directional differentiation and proliferation, which is the important point for eraly intervention of development and altering diabetes process.Autophagy is a authophagosome which is formed by the double part membrane package cytoplasm detached from no-ribosomes area in the rough endoplasmic reticulum and degradated organelles and proteins from the dells. Authophagosome is fuesed with lysosome to be autolysosome, whch can degrade the inner contents, ao as to complete the process of metabolism and regeneration of some organelles.Atg7 is an important regulator for maintaining multidirectional differentiation of adult stem cells.Methods:This study aimes to use the rice Ilet MIN6 cells as the model. different inhibitors and introducers for autophagy were added into In vitro culturation of MIN6 cells. The changes of ghrelin and Inssulin expressions were determined, which can reveal the changes of ε/β potentia. This study will provide a basis for immer mechanism of diabetes. MethodsIlet MIN6 cells were used as the model. Different inhibitors and introducers for autophagy were added into in vitro culturats of MIN6 cells. Western blot and rt-PCR were used to determine the key factors Includeign LC3, Atg7, LAMP2 and P62 exoressions on autophagy.Meanwhile, grelin and insulin insulinexpression distrubutions and changes were investigated by immunofluorescence, which can reveal the changes of ε/β potentia. Results:1. In the hungary cell group, Beclinl espression reduced (P<0.05), and the levels of Atg7 and LAMP-2 ecpression increased, Lc3 and P62 expressions had no changes; Beclinl and LC3 expression levels in the 3-MA group reduced (P<0.05) and no remarkable changes were found in the other genes. Western-blotting revealed that the P62 levels In the Inhibition group was higher than the control group (P<0.05), Lc3-Ⅱ/Ⅰ expression level decreased in the inhibition group (P<0.05); p62 level in the 3-MA group had no remarkable changes, and Lc3-Ⅱ/Ⅰ reduced remarkably (P<.05). LAMP-2 expression was similar among the three groups.2. The results of RT-PCR and immunofluorescence were similar. Insulin transcripting levels in the hungary cell groups increased significantly; insulin gene transcripting levels reduced in the 3-MA group; gene transcripting levels of ghrelin in the hungary cell group and 3-MA groups decreased remarkably.3. Immunofluorescence of cell differenciation revealed that Insulin secreting was higher In the hungary cell group than that in the control group; however, the Insulin level In the 3MA group reduced remarkably. Ghrelin secreting level was reduced in the hungary group and 3 MA group than the control group.Conclusions:1. Insulin gene transcripting increased remarkable when inducing authophagy; grelin reduced remarkably during authophgy, whichh revealed the imbalance of ε/β .ε/β imbalance may be related to autophagy.2. Autophagy was not successfully introduced in the present research based the detected levels of studied factors.