Research About Roles Of PI3K-Akt Signaling On Glucose Induced Lipogenesis In Goose Primary Hepatocytes

There are closely link between glucose metabolism and lipogenesis. When total energy intake exceeds energy expenditure, excess carbohydrates are converted to fatty acids and deposited for storage as triglycerides in hepatic and adipose tissue because metabolic product of glucose as acetyl CoA can provide substrate for fatty acid synthesis. Thereby, glycogen and triglycerides are broken down into glucose in the absence of glucose in body. PI3K-Akt signaling is a critical pathway involved in regulating glucose and lipid metabolism and is existed widely in vivo. However, whether the regulation of glucose on the liver steatosis has a potential link with PI3K-Akt signaling has not been reported. In this study, to illuminate effects of PI3K-Akt signaling inhibition on glucose-induced lipogenesis in goose primary hepatocytes, hepatocytes were cultured with the PI3K inhibitor LY294002 to inhibit the activition of PI3K-Akt signaling; then pre-treated with glucose (5,20,35μmol/L) for 24h to identify the impossible role of lipogenesis and activition of PI3K-Akt signaling. At the same time, under condition of PI3K-Akt signaling inhibition, hepatocytes were treated with glucose for 12h to identify whether glucose-induced lipogenesis was linked to the activition of PI3K-Akt signaling. The results of this study as following:1. Goose primary hepatocytes were pre-treated with LY294002 (10,20,40μmol/L) for 24h. Transcription level of key factors involved in PI3K-Akt signaling was examined using Real-time PCR. The data showed that treatment with LY294002 significantly decreased the transcription level (PI3K, Akt2, mTOR, raptor) compared with the control group; enzyme activities (PI3K, Akt1, mTOR, S6K) were inhibited; the protein levels of S6K and P-S6K were significantly decreased compared to control group. The above datas suggested that PI3K-Akt signaling and downstream target mTORCl were inhibited by LY294002.2. Goose primary hepatocytes were pre-treated with LY294002 (10,20,40μmol/L) for24h. Transcription levels of genes involved in lipogenesis were examined by Real-time PCR. The results showed that LY294002 down-regulated the mRNA level of genes as SREBP-1c, ChREBP, FAS, ACCa, LXRa, as well as the protein concertration of ACCa and FAS; the protein expression of ACCa was decreased compared with the control group. Accordingly, hepatocytes were pre-treated with LY294002 (10,40μmol/L) for 24h. Intracelluar lipid accumulation was examined using Oil red O staining, suggesting LY294002 treatment reduced intracelluar lipid accumulation compared to control group. The above results suggested that inhibiton of PI3K-Akt signaling down-regulated gene expression and protein level involved in lipid synthesis, leading to decrease lipid synthesis in hepatocytes.3. Hepatocytes were cultured with glucose (5,20,35μmol/L) for 24h. Translational levels of PI3K, Akt2, raptor, S6K were increased in a dose-dependent manner, as well as the protein concentration of PI3K, Akt2, mTOR, S6K. Accordingly, protein expression of S6K and P-S6K were increased compared to control group. The results suggested that glucose was able to stimulate PI3K-Akt signaling and downstream target mTORC1.4. Hepatocytes were cultured with glucose (5,20,35μmol/L) for 24h. The transcription levels of SREBP-1c, ChREBP, FAS, ACCa, LXRa were elevated; enzyme activities of FAS and ACCα and protein expression of ACCa were increased; hepatocytes were treated with glucose (5,35μmol/L) for 24h. Oil red O staining was used to examine intracelluar lipid accumulation. The data showed that treatment with glucose elevated lipid accumulation in a dose-dependent manner. Above results suggested that glucose stimulated de novo lipogenesis through stimulating the gene expression and protein level involved in lipogenesis in goose primary hepatocytes.5. Hepatocytes were pre-treated with LY294002 (10,20,40μmol/L) for 12h, then co-cultured with glucose (5,35νmol/L) for 12h. The results showed that LY294002 co-cultured with glucose significantly up-regulated the gene expression of PI3K, Akt2, mTOR, raptor compared with LY294002 treatment group, as well as protein concertration of PI3K, Aktl, mTOR, S6K. Cells co-cultured with glucose and LY294002 decreased the protein level of S6K and P-S6K compared with treatment group of glucose, The obove results suggested that glucose-dependent PI3K-Akt signaling can be attenuated by LY294002.6. Hepatocytes were pre-treated with LY294002 (20μmol/L) for 12h, then co-cultured with glucose (5,35μmol/L) for 12h. The results showed that LY294002 co-cultured with glucose significantly up-regulated gene expression of SREBP-1c, ChREBP, FAS, ACCa, LXRa compared with the LY294002 treatment group, as well as enzyme activities of FAS and ACCa. Cells co-cultured with glucose and LY294002 decreased the protein level of ACCa compared with glucose treatment group, suggesting glucose induced de novo lipogenesis via stimulating PI3K-Akt signaling in goose primary hepatocytes and the phenomenon can be attenuated by PI3K inhibitor LY294002.