The Protective Role Of ERp46in Palmitate-induced Endoplasmic Reticulum Stress As Well As The Intervention Of Exendin-4

Endoplasmic reticulum stress is proposed as one of the molecular mechanismsleading to insulin resistance and β-cells failure. ERp46(Endoplasmic reticulumprotein46), a new endoplasmic reticulum resident protein, is highly expressed inpancreatic tissue and has been proven to improve β-cells function in glucose toxicity.The researches in biological function of ERp46are still fewer. However, there havenot been any reports about the effect of ERp46in pancreatic β-cells lipotoxicity so far.In this study we tried to discuss whether ERp46was involved in palmitate-mediatedβ-cells toxic injury as well as the intervention of long-acting glucagon like-peptide-1(GLP-1) Exendin-4on pancreatic β-cells lipotoxicity and the expression of ERp46.We suggested that ERp46can be a new target in anti-diabetic prevention and therapyfield. This study is divided into three parts.Part Ⅰ: The harmful effect of palmitate on pancreatic β-cells as well as the alterexpression of ERp46and Insulin in TC6cellsThis section of the study aimed to observe saturated fatty acid palmitate on themouse insulinoma β-cell line βTC6cell proliferation as well as the expression ofERp46and insulin. CCK8analysis was used to detect βTC6cell proliferation. Theexpression levels of ERp46and insulin were measured by real-time PCR and Westernblot. Results showed that the cell proliferation were reduced and the expressions ofERp46and insulin were decreased in both dose-and time-dependent manners treatedwith palmitate. This indicated that ERp46was involved in palmitate-induced βTC6cells lipotoxic injury.Part Ⅱ: The protective effect of ERp46on palmitate-induced pancreatic βTC6cells lipotoxic injury To determine the possible function of ERp46on β-cells under high lipidcondition, we used siRNA method to knockdown ERp46gene expression in thissection of the study. CCK8analysis was used to detect βTC6cell proliferation. Theexpression levels of endoplasmic reticulum stress markers and insulin were measuredby real-time PCR and Western blot. Glucose-stimulated insulin secretion (GSIS) wasdetected by radio-immunity analysis. The alter of cell apoptosis was detected byAnnexinⅤ-PI and TUNEL analysis. Results showed that after the cells were treatedwith0.5mM palmitate for24h, cell proliferation reduced, endoplasmic reticulumstress markers ATF6α,PERK,IRE1α,JNK,CHOP,Caspase12increased, insulinbiosynthesis and GSIS decreased significantly as well as the cell apoptosis increasedcompared to NC alone. Under high concentration of palmitate, cell proliferationdecreased, the expression of ATF6α,PERK,IRE1α,JNK,CHOP,Caspase12increased, insulin biosynthesis and GSIS reduced and cell apoptosis increased inERp46siRNA compared to NC sample. This indicated the protective role of ERp46on improving β-cells function in βTC6lipotoxic injury.Part Ⅲ: Effect of Exendin-4on the expression of ERp46and endoplasmicreticulum stress markers in TC6cellsThis section of the study aimed to observe the protective function of Exendin-4on palmitate-induced pancreatic β-cells endoplasmic reticulum stress and theexpression of ERp46. The expression levels of ERp46and endoplasmic reticulumstress markers CHOP,Caspase12and P-JNK were measured by Western blot. Resultsshowed that ERp46level increased and CHOP, Caspase12, P-JNK levels decreasedgradually as the concentration of Exendin-4raised in dose-dependent manners withina certain range. The data demonstrated that Exendin-4protected pancreatic β-cellsfrom lipotoxicity by up-regulating the expression of ER chaperone ERp46andrelieving endoplasmic reticulum stress via three pathway.In conclusion, ERp46might alleviate palmitate-induced lipotoxic injury onβTC6cells and plays a protective role on improving β-cells function to some extent.Exendin-4might have a protect function via increasing ERp46level and viaalleviating three pathway of endoplasmic reticulum stress response.