Liraglutide Counteracts Palmitate-induced β-cell Apoptosis And Improves The Metabolism In ApoE^-/- Mice By Autophagy

BackgroundIt is all known that type2diabetes of which the pathogenesis including insulinresistance and relative impairment in insulin secretion has become hardly insolved. As weknown, β-cell apoptosis is the most important reason of disfunction of β-cell secretion.Therefore, how to protect β-cells has become the focus of this study. A series of clinicalstudies has confirmed that even if we controled blood glucose effectively, the function of βwould still gradually deteriorate. It is important to search for the mechanisms of β cellapoptosis and find a favorable treatment to delay the progress of type2diabetes andprotect β cell function. The glucagon-like peptide-1（GLP-1） can promote the secretion ofinsulin, increase the quantity of β cells and enhance their function，reduce the secretion ofglucagon, delay the gastric emptying, reduce postprandial hyperglycemia and lose weight,etc．But it is not clear the mechanism of protecting islet β cell. Studies have alreadyconfirmed that autophagy can make cells escape from apoptosis. As for degrading thelong-lived protein and the organelle, Autophagy is the unique pathway as we know. Recentstudies found that autophagy can prevent pancreatic β-cell apoptosis, maintain the quantityof β-cell and homeostasis. Based on all of the above, we suppose that liraglutide may playa role in reducing plamitate-induced β-cell apoptosis by activation of autophagy.ObjectiveBased on the theory we mentioned above, here we try to demonstrated thatliraglutide counteracts palmitate-induced β-cell apoptosis and protects ApoE^-/-mice byactivation of autophagy firstly. The purpose of this study is to clarify the role of autophagyin protecting of pancreatic islet β cells by liraglutide.Methods1. Liraglutide counteracts palmitate-induced β-cell apoptosis by activation of autophagyThe effects of liraglutide on palmitate-induced β-cell apoptosis. By applicating MTTreduction assay, we detected the death of β-cell influenced by liraglutide and palmitate. Byusing AnnexinV/PI double staining, we detected the apoptosis of INS-1cells.As for detecting the activation of β-cell autophagy. We use GFP-LC3plasmid andtransfect it into islet β-cells then observe the formation of punctate fluorescence by microscope. We also detected LC3B、 ATG7、Beclin1、p62、 ATG5protein expression,and observed autophagosome in the islet β-cell induced by liraglutide.The mechanism of liraglutide activating the autophagy in β-cell. Testing proteins andgene expression associated with autophagy and endoplasmic reticulum stress（ERS）,suchasGRP78、CHOP、JNK.To further clarify the role of autophagy in protecting β cell function by liraglutide. Wechose the autophagy inhibitor （3-MA, chloroquine） to pretreat pancreatic β cells, and thenobserved the protective effect on β cells by liraglutide. We still apply MTT assay andAnnexinV/PI double staining to see the apoptosis of pancreatic β cells including INS-1cell line.2. Liraglutide protects ApoE^-/-mice with high-fat diet by activation of autophagy.ApoE^-/-mice aged6weeks divided into4groups radomly,including group of normaldiet（A:N group）,high-fat die（tB:HF group）, high-fat diet and liraglutide injection（C:HF+Lgroup）, high-fat diet with liraglutide and chloroquine （CQ） injection（D:HF+L+CQgroup）.These mice fed high-fat diet12weeks and injected drugs4weeks.After16weeks,the levels of serum glucose，insulin，TC，TG，LDL and free fatty acids were tested. Thenthe ApoE^-/-mice were executed and frozen sections of their hearts，livers，kidney，hypothalamus， pancreas and fat were made. Hematoxylin and eosin staining andimmunohistochemisty were used to detected kinds of proteins.3. Liraglutide protects ApoE^-/-diabetic mice with high-fat diet by activation of autophagy.ApoE^-/-mice aged6weeks divided into4groups radomly,including group of normaldiet（A:N group）,high-fat die（tB:HF group）, high-fat diet and liraglutide injection（C:HF+Lgroup）, high-fat diet with liraglutide and chloroquine （CQ） injection（D:HF+L+CQgroup）.These mice fed high-fat diet8weeks and injected STZ into diabetic model. Andthen injected drugs30days. After more than13weeks, the levels of serum glucose，insulin，TC，TG，LDL and free fatty acids were tested. And the mice were executedimmediately, then we also collected frozen sections of heart， liver， kidney，hypothalamus, pancreas and fat. Hematoxylin and eosin staining andimmunohistochemisty were used to detected kinds of proteins.Results:1. Liraglutide counteracts palmitate-induced β-cell apoptosis by activation of autophagy.The apoptosis of Liraglutide plus palmitate group is higher than the group of palmitate-induced β-cel（lP＜0.05）. GFP-LC3transfected islet β-cells and we observed theformation of punctate fluorescence with fluorescence microscope; LC3B proteinexpression had been detected, p62had been increased，ATG7、Beclin1had beendecreased and we also observed autophagosome in the islet β-cell induced by liraglutidethough electron microscope. We determined the mechanism of liraglutide activating wasautophagy reacting ERS in pancreatic β-cell. Liraglutide increased islet β-cellthough,decreasing GRP78、CHOP. Pretreated autophagy inhibitor （3-MA, chloroquine） topancreatic β cells, liraglutide+palmitate+CQ/3-MA group’s apoptosis was higher than thegroup of liraglutide+palmitate-induced β-cell（P＜0.05）.2.Liraglutide protects ApoE^-/-mice with high-fat diet by activation of autophagy.After12weeks, the indexes of FFA and LDL-C on ApoE^-/-mice fed with high-fat dietwas higher than that in normal group（P＜0.05）. Medication4weeks，The levels of LDL-C and FFA of B group were higher than that in C group（P＜0.05）.The indexes of blood inB group had no difference with that in D group（P＞0.05）. The foam cell aggregation inheart and fat disposition in liver and kidney had been reduced of group C though lightmicroscope.The result of immunohistochemisty,group C LC3B expressed strongly in thepancreasand JNK3、CHOP decreased in the hypothalamus.3.Liraglutide protects ApoE^-/-diabetic mice with high-fat diet by activation of autophagy.After8weeks, the indexes of FBG、TC、LDL-C on ApoE^-/-mice fed with high-fat dietwas higher than that in normal group（P＜0.05）. Medication30days，The levels of bloodglucose, INS, TG, TC and LDL-C of B group were higher than that in C group（P＜0.05）.The foam cell aggregation in heart and fat disposition in liver and kidney had been reducedof group C though light microscope. The result of immunohistochemisty,group C LC3Bexpressed strongly in the pancreasand, JNK3decreased in the hypothalamus.ConclusionLiraglutide could prevent islet β-cell apoptosis induced by free fatty acid，and activateβ-cell autophagy, which may play the role through ERS pathway. However, by inhibitingautophagy, the prevention of apoptosis in high-fat induced islet β-cell treated withLiraglutide disappeared. Liraglutide effectively improved the levels of blood glucose,insulin, lipids and also reduced fat disposition in liver and foam cell aggregation in aortasof high-fat diet mice. In conclusion, we indicated that Liraglutide could counteractspalmitate-induced β-cell apoptosis and improves the metabolism in ApoE^-/- mice with high-fat diet by activation of autophagy.