The Research Of Endoplasmic Reticulum Stress Regulating Lipid Metabolism In Goose Hepatocytes Via PI3K-Akt-mTOR Activation

Endoplasmic reticulum stress can induce rodents and human hepatic steatosis models have been reported. at the same time, recent studies have shown that the endoplasmic reticulum stress plays an important role in the regulation of fat deposition in the liver. Endoplasmic reticulum stress activates the proteolytic cleavage of the lipogenic transcription factor sterol regulatory element binding protein-1c leading to the induction of lipogenic enzyme expression. Endoplasmic reticulum stress has associated with impaired VLDL secretion by inhibiting apoB100 secretion. But the role of PI3K-Akt-mTOR signaling pathway on endoplasmic reticulum stress in lipid metabolism few research. In this study, after the inhibitors (LY294002?NVP-BEZ235?rapamycin) of PI3K-Akt-mTOR pathways signal treatment in vitro culture of goose the original generation of liver cells and then tunicamycin-induced endoplasmic reticulum stress, using the fluorescent quantitative PCR method, western blotting protein assay. Elisa kit test and oil red O staining method to detect farty acid synthesis, fatty acid oxidation in VLDL-TG secretion of transshipment and related indicators to reflect the change of lipid metabolism, analysis of endoplasmic reticulum stress effects on the liver lipid metabolism and mediation of PI3K-Akt-mTOR signaling pathway, in order to explore PI3K-Akt-mTOR signaling pathways and endoplasmic reticulum stress on molecular mechanism of lipid metabolism in the liver and provide theoretical basis for the level of gene regulation. The findings are as follows:1. Goose primary'hepatocytes were pre-treated with tunicamycin (0.0.2,2.20 ?mol/L) for 24 h. The data showed that treatment with tunicamycin significantly increased the transcription level of key genes involved in endoplasmic reticulum stress (BIP, EIF2a, ATF6?XBP1) compared with the control group; significantly increased concentration of endoplasmic reticulum stress key protein BIP/GRP78 protein. The above datas suggested that ER stress was activated by tunicamycin in Goose primary hepatocytes.2. After the hepatocytes treatment with tunicamycin, the results showed that compared with the control group, treatment with tunicamycin decreased gene mRNA expression of mTORcl and inecreased gene mRNA expression of PI3K, S6K(P<0.05); the gene mRNA expression of Aktl was not significant. Meanwhile, enzyme activities of PI3K, mTOR, S6K, compared with the control group, the protein expression of mTOR content decreased, while PI3K and protein S6K activity increased, indicating that tunicamycin-induced endoplasmic reticulum stress can regulate P13K and mTORc1 genes.3. After the hepatocytes combined treatment with PI3K-Akt-mTOR pathway inhibitors and tunicamycin, The results showed that LY294002, NVP-BEZ235, and rapamycin co-cultured with tunicamycin significantly down-regulated the gene expression of BIP, EIF2a, ATF6, and XBP1 compared with the tunicamycin control group(P<0.05), as well as protein concertration of BIP/GRP78 (P<0.05). NVP-BEZ235, Rapamycin significantly higher LY294002 inhibition effect (P<0.05), the difference between NVP-BEZ235 and Rapamycin was not significant, our results strongly indicate that LY294002, NVP-BEZ235, and rapamycin might down-regulate the tunicamycin-induced endoplasmic reticulum stress in primary goose hepatocytes.4. After the hepatocytes treatment with tunicamycin, the results showed that the contents of intracellular TG display was higher than the control group, and reduced intracellular VLDL content; At the same time, oil red O staining cell lipid drops granules increase, increase lipid deposition. FAS?ACCa?PPARy?PPARa?CPT1? MTP?ApoB?DGAT1?DGAT2 genes mRNA expression compared with the control group significantly reduced, SREBP-1 expression increased, among them (P< 0.05), the ACOX1 gene mRNA expression no significant difference (P>0.05). decreased enzyme activities of FAS, CPT and MTP (P<0.05), the kinase expression activities of ACC no significant difference. Western blot test CPT1 protein content after tunicamycin-induced ER stress showed that CPT1 protein expression levels significantly lower than the control group. These results indicate that the goose liver cells ER stress can involved in cell lipid metabolism by regulating the cellular fatty acid synthesis, oxidation and lipid transport the expression of related genes.5. After the hepatocytes combined treatment with PI3K-Akt-mTOR pathway inhibitors and tunicamycin, The results showed that compared with the tunicamycin control group the intracellular TG content decreases, extracellular TG content no change,intracellular VLDL content increase. While Oil red O staining showed intracellular lipid droplets particles decreased significantly, ACCa?FAS?SREBP-1?CPTl?ACOX1?MTP?ApoB?PPARy and DGAT2 genes expression decreased, PPARa and DGAT1 genes mRNA expression increased compared with the tunicamycin control group (P<0.05). ACC protein concentration decreased, FAS protein concentration difference was not significant; increased protein concentration of CPT1, MTP (P<0.05), decreased protein concentration of ACC (P<0.05), the protein concentration of of FAS no significant difference. Western blot test CPTl protein content showed that decreased the protein expression of CPT1. These results show that when the PI3K-AKT-mTOR pathway was inhibited, can effectively mitigate the effects of endoplasmic reticulum stress on lipid metabolism processes.