The Signaling Mechanism Of NEFAs Inducing Insulin Resistance Through Endoplasmic Reticulum Stress In Hepatocytes Of Dairy Cows

In cows transition, because of the dry matter intake reduced and increased energy demand, dairy cows often in a state of energy negative balance(NEB). The NEB induce the body fat mobilization and makes a mass of non-esterified fatty acids(NEFAs) release into the blood, leading to high concentration blood NEFAs. High concentration NEFAs is a crucial factor of cows fatty liver, ketosis and inflammatory diseases. However, The mechanism of insulin resistance in NEB cows liver is not clear. Recently, There have studies indicated that endoplasmic reticulum stress(ERS) has close connection with the insulin resistance.However, the relationship between ERS, hepatic insulin resistance and high NEFAs is unclear. Therefore, to explore the mechanism of high concentration NEFAs induces hepatic insulin resistance in severe NEB dairy cows, this study detect the NEB dairy cows in vivo and culture calf hepatocytes in vitro.The concentrations of NEFAs, BHBA, and indexes of glucose metabolic and liver damage were determined in the NEB cows and healthy cows. The results suggested that there are serious energy metabolic disorders, insulin resistance and liver damage in NEB cows. The ERS and insulin pathway key molecules were determined in the NEB cows liver. The phosphorylation level of PERK and IRE1α and protein level of cleaved-ATF6 were significantly higher in the NEB cows than in the healthy cows. Moreover, the phosphorylation levels of IRS1 was significantly increased and AKT and GSK3β were significantly reduced in NEB cows. Taken together, these results indicate that NEB cows suffer ERS and insulin resistance in liver.To explore the effects of NEFAs on ERS in hepatocytes, hepatocytes were incubated with 1.2m M NEFAs for time course test. The result showed that PERK, IRE1α, cleaved-ATF6 were activated with the increase of NEFAs processing time. In addition, the m RNA levels of ERS-associated molecules ATF4, P58 IPK, ATF6, XBP-1S were increased significantly. Hepatocytes were treated with different concentrations of NEFAs(0, 0.6, 1.2 and 2.4m M) and inhibitor of ERS(TUDCA). The results showed that NEFAs can induce ERS, and TUDCA can alleviate NEFAs-induced ERS in hepatocytes.Ultrastructural analysis of hepatocytes showed that NEFAs exposure led to dilatation in endoplasmic reticulum. Hepatocytes were treated with different concentrations of NEFAs and TUDCA, then treated with insulin. The phosphorylation level of IRS1, AKT, GSK3β and the m RNA levels of downstream molecules PEPCK, G6-pase were determined. Take together, the results showed the NEFAs can induce insulin resistance, and TUDCA can alleviate NEFAs-induced insulin resistance in calf hepatocytes.To determine the effects of IRE1α, PERK, ATF6 pathways on NEFAs-induced insulin resistance in calf hepatocytes. We treated the cells with sh IRE1α, GSK2656157, AEBSF to inhibit IRE1α, PERK, ATF6 pathways respectively. The results showed that the interference suppression IRE1α and PERK can significantly relieve NEFAs-induced insulin resistance.To further explore the mechanism of IRE1α mediated NEFAs-induced insulin resistance, we evaluated the effects of JNK on NEFAs-induced insulin resistance by treatment the cells with JNK inhibitor. The results suggested that inhibition of JNK activity can significantly improve NEFAs-induced insulin sensitivity in calf hepatocytes. The results showed that the interference IRE1α can significantly reduce NEFAs-induced JNK activation and expression of downstreaminflammatory cytokines.In conclusion, These results suggest that the presence of ERS and insulin resistance in NEB cows liver. NEFAs induce calf primary hepatocytes ERS and insulin resistance. Moreover, ERS mediates NEFAs-induced insulin resistance. IRE1α mediates NEFAs-induced insulin resistance in calf hepatocytes through JNK.